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Afro refuses to wear the No. 1 headband as the rules require, and spends his days making wooden sculptures of people from his past instead of fighting. One dark and stormy night, Jinno (now mindlessly taking orders) and a mysterious woman named Sio attack Afro. Jinno easily defeats him and takes the No. 1 headband for Sio and then robs his father's grave of his remains. Sio is revealed to be Jinno's sister. Sio tells Afro her plans to resurrect Afro's father, Rokutaro, so she can torture him as revenge against Afro for the lives he has destroyed, including her brother's and her own. Sio challenges Afro to find the No. 2 headband to earn the right to challenge her. Afro, determined to recover the No. 1 headband and his father's remains, sets off to find the No. 2 headband and eventually learns that the bearer of the No. 2 headband is a man named Shichigoro.
Afro, in search of Shichigoro, coincidentally kills the kidnapper of Schichigoro's son and prompts Shichigoro to buy him a drink. They eventually fight to the death with the No. 2 headband at stake, and upon victory Afro continues on toward Sio. Along the way he is observed by three masked, android warriors from his past. The warriors are revealed to be a part of Sio's original plan to overwhelm Afro in the final battle, but apparently had decided to battle Afro early in an attempt to finish him off before Sio had to dirty her hands.
Afro battles and defeats the three while Sio attempts to resurrect a mind-controlled version of Afro's father. However, Rokutaro is revived before he is completely restored, leaving him merely a mindless, though apparently somewhat controllable, killing machine. Afro defeats the three and is soon confronted by Sio, Jinno, and his resurrected father. With Afro hesitant to fight his father, Rokutaro defeats and kills him (stops his heart from beating). Jinno comes to Afro's aid, though pointless as Sio points out, in a sudden flash of selective memories of being a childhood friend and sparring partner with Afro. Landing only a few blows, Jinno is quickly killed by Rokutaro, who kills Sio in the same blow when she attempts to come to Jinno's aid. The cybernetic remains of Jinno give off an electrical surge, which, conducted through the spilled blood of Sio, restarts Afro's heart. Afro apparently defeats Rokotaro and emerges from the scene wearing the No. 1 headband.
Afro walks away from the battle, wearing the No. 1 headband and clutching the No. 2 headband in right hand. He comes across Shichigoro's orphaned son, who had been following him, and hands him the No. 2, telling him to challenge him when he's ready.
In an Associated Press interview in 2007, Takashi Okazaki confirmed there would be a sequel to the anime series, and that it would also be shown on Spike TV. In 2008, the sequel was announced to be a TV movie titled Afro Samurai: Resurrection, and that actors Lucy Liu and Mark Hamill would join the voice acting cast. Hip hop artist RZA also came back to provide the soundtrack for the movie. Afro Samurai: Resurrection debuted on Spike TV on the night of January 25, 2009. On July 16, 2009, Afro Samurai: Resurrection was nominated for an Emmy in the "Outstanding Animated Program (for programming one hour or more)" category in the 61st Primetime Emmy Awards and the Creative Arts Emmy Awards. At the Emmy awards, Afro Samurai: Resurrection lost to Destination Imagination, a TV movie based on Foster's Home for Imaginary Friends. The art director of Afro Samurai: Resurrection, Shigemi Ikeda, won an Emmy for his work on Resurrection, which is the first ever awarded for work on a Japanese-animated production. Afro Samurai: Resurrection was the first Japanese anime to be nominated for and win an Emmy.
Late 2009 also saw the release of Afro Samurai: Complete Murder Sessions on Blu-Ray and DVD. A 4-disc collection of both Afro Samurai Director's Cut and Afro Samurai: Resurrection, together in one complete boxset.
Announced at the 2006 Comic-Con, a live action version of Afro Samurai was said to be in the making. On 2011-07-21, Gonzo K.K. and Indomina group announced Indomina group had obtained the rights to produce the film, with Samual L. Jackson, Jasbinder Singh Mann (Indomina Group Vice Chairman and CEO), Shin Ishikawa (Gonzo Studios) as producers; Eli Selden of Anonymous Content as executive producer.