Guys and Dolls JR. opens with a bustling street scene alive with Times Square characters. Some gamblers enter and trade tips about different horses that they are considering placing bets on from the daily scratch sheet ("Fugue for Tinhorns"). As the gamblers finish their pitch, Miss Sarah Brown and the Mission Band enter, playing a hymn ("Follow the Fold"). She warns the gamblers of the evils of their ways, but her sermon falls on deaf ears, so she and the band exit dejectedly. Lt. Brannigan, of the New York Police Department, enters and warns the gamblers not to try to organize their crap game. Nathan Detroit enters and, after Brannigan exits, complains that there is nowhere for the crap game to take place unless he can come up with $1000 to rent the Biltmore Garage. Nathan, Benny, Nicely and the gamblers sing of their frustration in  "The Oldest Established." Sky Masterson is rumored to be in town, and Nathan tries to think up a bet to place with Sky that he cannot lose to come up with the money for the game. Meanwhile, Adelaide enters with the Hot Box Girls and gives Nathan his anniversary present, marking their fourteenth year of engagement!) She also warns him not to try to organize his crap game and then exits as Sky Masterson enters. Nathan has instructed his boys to get the lowdown on how much cheesecake and how much strudel is sold at a popular restaurant. With the advance information, Nathan attempts to sucker Sky into a bet for $1000, but Sky relates a story his father told him, and refuses. So, Nathan counters with another bet: that Sky can't take a specified woman on a trip to Havana. A confident Sky takes the bait, and Nathan names the missionary, Miss Sarah Brown, as the woman. Only then does Sky realize the difficulty he is facing, and Scene 1 ends.

Sky goes to the Save-a-Soul Mission to get a date with Miss Sarah Brown. He and Sarah discuss the lack of sinners in the mission, and Sky proposes a trade: he will personally guarantee twelve sinners for the struggling mission if Sarah will accompany him to Havana for dinner. She refuses, saying that the man she will love will not be a gambler, so Sky asks her to describe the man of her dreams. She replies that she'll know when the right man comes along ("I'll Know"). Their song ends in a kiss that quickly turns into a slap in the face for Sky.

At the Hot Box, Adelaide and the Hot Box Girls perform a number ("A Bushel and a Peck"). After the show, Adelaide announces to Nathan that it is time they finally got married, warning him again not to start his crap game back up.  Nathan exits hurriedly to do just that, and she sings of her frustration ("Adelaide's Lament").

Benny and Nicely have been watching Sky follow Sarah and the Mission band, hoping he will lose his bet with Nathan, and the $1000 windfall will allow them to hold the crap game. They observe that men all over the world have a weakness for falling in love ("Guys and Dolls").

Sarah and the band return to the mission. Arvide Abernathy, the bass drum player and Sarah's grandfather, encourages her to pay some attention to Sky. General Cartwright, the head of the Save-a-Soul Mission, arrives and explains that the Broadway mission's poor performance in attracting sinners is forcing her to close the branch. Sky appears and protests the close of the mission, reminding Sarah of his IOU for one dozen sinners. Desperate, she accepts and then guarantees the General that there will be one dozen genuine sinners in the mission the following evening.

All the crap shooters, including Big Jule (a very tough, gun-toting gangster from Chicago) are wearing red carnations as their badge of entry for the game, which still has no location. Lt. Brannigan appears, notices all the red carnations and asks Nathan what's going on. Benny sees Adelaide with some of the other hot box dancers and covers for Nathan, telling Brannigan that the carnations are for guests of Nathan's bachelor party. Adelaide hears this and excitedly tells Nathan that they will elope the following evening after the show. As Adelaide exits, Nathan tells Benny that he still has not received any money from Sky, so they worry that Miss Sarah actually went to Havana.

In fact, Sarah and Sky are in Havana at that very moment. After a "dulce de leche" drink, Sarah tells Sky how she feels ("If I Were a Bell"). Realizing that he is falling in love with her, a guilty Sky tells Sarah about the bet that he made with Nathan. She reluctantly allows him to take her back to New York.

Outside the Mission at 4:00am. Sarah and Sky run into Adelaide, who is returning from a bridal shower. Sky reveals to Sarah that his real name is Obediah. Sky and Sarah sing to each other of their new found love ("I've Never Been in Love Before"). At the end of the song they are met by Arvide, who is returning from a night of mission work. Police bells sound, and several gamblers suddenly flee the mission and the grasp of Lt. Brannigan. Nathan has held the crap game in the Mission. Sarah is convinced Sky's trip to Havana was part of Nathan's plan all along, so she angrily breaks it off with Sky.

Adelaide and the girls perform another number at the Hot Box ("Take Back Your Mink"). Sky enters and bumps into Nicely, who is looking for Adelaide. He has been sent to tell her that Nathan cannot meet her after the show as planned. Nicely tells Sky that Nathan is still at the game because Big Jule won't allow the game to end until he wins back all the money he lost. Adelaide enters, realizes that Nathan is still running the crap game and tells Sky to tell Nathan that she never wants to see him again. ("Adelaide's Second Lament").

Sarah, angry that she is in love with Sky and convinced that he helped set up the mission crap game, tells Arvide that she wants to leave. Arvide sings to her, "More I Cannot Wish You". Sky and Nicely pass by on their way to the crap game, and Sky tells Sarah and Arvide that he intends to honor his IOU. He and Nicely open a manhole cover and descend to the sewers, where the crap game is being held.

The game is proceeding furiously ("The Crapshooters' Dance"). At the end of the dance, Big Jule and his gun remind the fatigued group that they will all stay and play until he wins his lost money back. Sky enters as Big Jule begins fixing the game against Nathan. He proposes to bet $1000 against each player on one roll and, if he wins, each player must go to the mission. As he prepares to roll, he prays to Lady Luck for help ("Luck Be a Lady").

Nathan runs into Adelaide, who makes him squirm to patch things up, until Nicely and Benny arrive to remind him of his obligation to go to the prayer meeting at the Mission. Adelaide, of course, is convinced that Nathan is just lying again. But, at a few minutes past midnight, all the gamblers enter the mission to the surprise of Sarah and the delight of General Abernathy. The gamblers are compelled to testify to their sins, and several do, leading up to Nicely's musical testimony ("Sit Down, You're Rockin' the Boat"). After the song, Nathan tells Sarah and the others about his bet, confessing that Sky claimed to have lost, but Sarah is confused because she knows that Sky actually won the bet. She exits as the General leads the gamblers in a hymn ("Follow the Fold").

Sarah and Adelaide meet on the street early in the morning and commiserate about Sky and Nathan ("Marry the Man Today").

Adelaide appears in a wedding gown and calls for Nathan. Nathan tells her that he hasn't found a place for them to get married. The Mission Band enters, led by Sky and Sarah. Nathan asks Sky if he can get married in the Mission. Arvide, who has already married Sky and Sarah in the mission earlier, happily offers to do the same for Adelaide and Nathan. All is well on Broadway as the curtain falls.