1932, Clarksdale, Mississippi. Twin brothers and World War I veterans Elijah "Smoke" and Elias "Stack" Moore return home after seven years working for the Chicago mob. Using stolen mob money, they buy an abandoned sawmill from the landowner Hogwood and plan to open a juke joint for the local Black community. Their cousin Sammie, a gifted young blues guitarist, joins them despite warnings from his pastor father Jedidiah, who says blues music is sinful.

The brothers build their team: blues legend Delta Slim, the shopkeeper couple Grace and Bo Chow, the field worker Cornbread as bouncer, and Smoke's estranged wife Annie, a Hoodoo practitioner, as cook. Smoke and Annie are still broken by the death of their infant daughter. Stack reconnects with Mary, the white-passing ex-girlfriend he abandoned years earlier to protect her.

Nearby, a pale Irish stranger named Remmick hides from Choctaw vampire hunters by sheltering with a married couple of Klansmen, whom he turns into his first thralls.

On opening night, Sammie, Delta Slim, and the singer Pearline perform - and Sammie's playing is so powerful it summons the spirits of musicians past and future. The music draws Remmick and his growing pack to the juke joint. They offer money and music in exchange for being let in. Smoke, suspicious, refuses. When Mary steps outside and speaks with Remmick, he turns her, and she seduces and fatally bites Stack. Smoke shoots her, but she is unharmed and escapes. Cornbread and the departing patrons are turned as well.

Stack rises as a vampire but flees when Annie repels him with pickled garlic juice. Annie realizes what they are facing and arms the survivors with Hoodoo knowledge. Trapped by the invitation rule, Remmick tries bargains and threats. He promises to spare everyone in exchange for Sammie, whose gift could summon the lost community he misses. He also reveals that Hogwood leads the local Klan and plans to attack the joint at dawn. When Remmick threatens the Chows' daughter Lisa, Grace snaps and invites the vampires in herself, choosing to fight rather than hide.

The night becomes a massacre. Grace, Bo, Annie, and Delta Slim are killed. Remmick turns Pearline. Smoke knocks out his own turned brother rather than kill him, then helps Sammie bring down Remmick, who burns with his horde in the sunrise. Smoke sends Sammie home, then kills Hogwood and his Klan men at dawn, dying from his wounds. In death, he is reunited with Annie and their daughter. Sammie ignores his father's pleas and leaves for Chicago to make music.

The epilogue jumps to 1992. Sammie, now an old blues legend played by Buddy Guy, is visited at his club by Stack and Mary, who have not aged. Stack explains that Smoke spared him on the condition he never harm Sammie. They offer to turn the dying Sammie so he can keep playing forever. Sammie declines - his life was enough. They ask him to play one more song, and as they listen, Sammie says that the day they all opened the juke joint was the greatest day of his life, until the sun went down. Stack agrees softly: it was the last time he ever saw Smoke, or the sun - and the only time they were all truly free.

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