Revenge. That's all that the man named Afro knows. His father was murdered when he was just a small child, killed by a man seeking the power of The Number One, a headband said to bestow the power of a god. Only he who carries The Number Two has the right to challenge The Number One, and to avenge his father, Afro has dedicated his life to becoming The Number Two and killing the man who wears The Number One.

Afro Samurai is a third-person action game based on the anime created by Takashi Okazaki and Gonzo and starring Samuel L. Jackson. Each stage chronicles an important event in Afro's quest for revenge, culminating in a boss fight at the end. The defining feature of the combat is Afro's ability to enter "focus mode," where the screen goes black-and-white and the action slows down, allowing for a "perfect slice" attack which can slice enemies literally to pieces. It must be charged after use, with the charge represented by a pendant hanging from Afro's sword.

As Afro kills enemies, he levels up earning new skills and increasing his power and stamina. Collecting "mementos" strewn around each stage will also unlock special abilities called "father's legacy", which are often extra-devastating moves that can easily take down even stronger enemies.