In 1941, SS-Standartenführer Hans Landa interrogates French dairy farmer Perrier LaPadite in his home as to the whereabouts of the last unaccounted-for Jewish family in the area, the Dreyfus family. Landa suspects that the LaPadites are hiding the Dreyfuses under their floorboards and, in exchange for the Germans agreeing to leave his family alone for the rest of the war, LaPadite reluctantly confirms it. Landa orders his SS soldiers to shoot through the floorboards, killing all but one of the Dreyfus family; Shosanna, the daughter, escapes. As she runs, Landa decides to spare her.
In 1944, Lieutenant Aldo Raine of the First Special Service Force rounds up and recruits Jewish-American soldiers to the Basterds, a commando unit formed to instill fear among the German soldiers by killing and scalping them. The Basterds include Donny "The Bear Jew" Donowitz and Sergeant Hugo Stiglitz, a rogue German soldier who murdered thirteen Gestapo officers. In Germany, Adolf Hitler interviews a young German soldier, Private Butz, the only survivor of a Basterd attack on his squad, who reveals that Raine carved a swastika into Butz's forehead with a bowie knife so that in the future, Butz could never hide the fact that he was once a Nazi soldier.
Shosanna Dreyfus is living in Paris, operating a cinema under the name "Emmanuelle Mimieux." She meets Fredrick Zoller, a German sniper famed for killing 250 Allied soldiers in a single battle. Zoller stars in a Nazi propaganda film, Stolz der Nation (Nation's Pride). Infatuated with Shosanna, Zoller convinces Joseph Goebbels to hold the film's premiere at her cinema. Landa, who is the head of security for the premiere, interrogates Shosanna, hinting that he suspects her real identity. Shosanna plots with her Afro-French lover and projectionist, Marcel, to set the cinema ablaze during the premiere and kill the Nazi leaders who will attend.
Meanwhile, British Commando Lieutenant Archie Hicox is recruited to lead Operation Kino, a British plan to attack the premiere in cooperation with the Basterds. Hicox, along with Basterds Stiglitz and Wilhelm Wicki, goes to a basement tavern in German-occupied northern France to meet with German film star Bridget von Hammersmark, an undercover Allied agent who will be attending the premiere in Paris. Hicox inadvertently draws the attention of Wehrmacht Sergeant Wilhelm and Gestapo Major Dieter Hellström with his unusual accent and mannerisms; he ultimately gives himself away by using a British hand gesture; indicating his request for 3 whiskeys from the bartender by raising his index, middle, and ring finger, rather than the German method of raising the index, middle, and thumb. Their covers blown, Stiglitz and Hicox shoot Hellström, triggering a gunfight that kills everyone in the tavern except Sergeant Wilhelm and von Hammersmark, who is shot in the leg. Raine arrives and negotiates with Wilhelm for von Hammersmark's release, but she shoots Wilhelm when he lowers his guard. Raine, believing von Hammersmark set Hicox and his men up, tortures von Hammersmark, who convinces him that she is loyal to the Allies and reveals that Hitler will also be attending the film premiere. Raine decides to continue the mission. Later, Landa investigates the aftermath at the tavern and finds von Hammersmark's shoes and a napkin with her signature.
At the premiere of Stolz der Nation, Omar Ulmer, Donny, and Raine attend the premier with timed explosives strapped to their ankles. To hide their inability to speak German, they pose as Italian guests of von Hammersmark. However, Landa is fluent in Italian, holding a brief, tense conversation with the Basterds before allowing Donowitz and Ulmer to take their seats. Landa takes von Hammersmark to a private room, verifies that the shoe from the tavern fits her, and strangles her. Raine and another Basterd, Smithson "The Little Man" Utivich, are taken prisoner. Still, Landa has Raine contact his superior with the OSS and cut a deal: he will allow the mission to proceed in exchange for Landa's safe passage through the Allied lines, a full pardon, and various benefits after the war's conditionally inevitable end.
During the screening, Zoller slips away to the projection room and attempts to force himself on Shosanna. She pretends to acquiesce, then pulls a pistol and shoots him. Zoller, mortally wounded, manages to shoot and kill her before he dies. As Stolz der Nation reaches its climax, Shosanna's spliced-in footage tells the audience in English that they are about to be killed by a Jew. Having locked the doors of the cinema, Marcel ignites a huge pile of flammable nitrate film behind the screen as Shosanna's image laughs and the theater goes up in flames. Ulmer and Donowitz break into the opera box containing Hitler and Goebbels, submachine-gunning them both to death then firing into the crowd until the bombs go off, killing everyone in the theater.
Landa and his radio operator drive Raine and Utivich into Allied territory, where they surrender themselves to the remaining Basterds. Raine, apparently not according to plan, shoots the radio operator before ordering Utivich to scalp him. Despite his superiors having agreed to Landa's deal, Raine has him restrained and carves a swastika into his forehead, professing it to be his "masterpiece."
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