- Tom Cruise as Captain Nathan Algren, a Civil War and Indian War veteran. Although he is an exceptionally talented soldier, he is haunted by his role in the massacre of Native Americans at the Sand Creek or Chivington and/or the massacre of Native Americans at the Washita River. Following his discharge from the United States Army, he agrees to help the new Meiji Restoration government train its first Western-style conscript army for a significant sum of money. During the army's first battle he is captured by the samurai Katsumoto and taken to the village of Katsumoto's son, where he soon becomes intrigued with the way of the samurai and decides to join them in their cause. His journal entries reveal his impressions about traditional Japanese culture, which almost immediately evolve into unrestrained admiration of Japan.
- Ken Watanabe as Lord Moritsugu Katsumoto, the eponymous "Last Samurai," a former daimyo who was once Emperor Meiji's most trusted teacher. His displeasure with the influence of Omura and other Western reformers on the Emperor lead him to organize his fellow samurai in a revolt, which he hopes will convince the government not to destroy the samurai's place in Japanese society. Katsumoto is based on real-life samurai Saigō Takamori, who led the Satsuma Rebellion.
- Koyuki as Taka Katsumoto, widow of a samurai slain by Nathan Algren and younger sister of Moritsugu. She and Algren develop feelings for each other, and she gives him her husband's armor to wear in the final battle of the rebellion.
- Timothy Spall as Simon Graham, a British photographer and scholar hired as an interpreter for Captain Algren and his non-English speaking soldiers. Initially portrayed as a friendly yet mission-oriented and practical-minded companion, he later comes to sympathize with the samurai cause and helps Algren rescue Katsumoto from Imperial soldiers. Graham is loosely based on real-life British Japanologist Ernest Mason Satow, who befriended Saigō Takamori during the Satsuma rebellion.
- Shin Koyamada as Moritsugu Nobutada, Katsumoto's only son and lord of the village where Algren is sent. Nobutada befriends Algren when Katsumoto assigns him to teach Algren Japanese culture and the Japanese language. He dies when he willingly chooses to distract Imperial troops so his father can escape their custody.
- Tony Goldwyn as Colonel Bagley, Nathan Algren's former commanding officer in the 7th Cavalry Regiment. Ruthless and amoral, Bagley convinces Algren to serve as a training instructor for the Imperial Army despite Algren's hatred of Bagley for his role in the Washita River massacre. In contrast to Algren, Bagley is arrogant and dismissive of the samurai, at one point referring to them as nothing more than "savages with bows and arrows". He is killed by Algren who throws a sword into his chest when Bagley tries to shoot Katsumoto in the final battle.
- Masato Harada as Matsue Omura, an industrialist and pro-reform politician. He quickly imports Westernization and modernization while making money for himself through his ownership of Japan's railroads. Coming from a merchant family, a social class repressed during the days of Shogun rule, Omura openly expresses his contempt for the samurai and takes advantage of Emperor Meiji's youth to become his chief advisor, persuading him to form a Western-style army for the sole purpose of wiping out Katsumoto and his rebels while ignoring their grievances. His appearance is designed to evoke the image of Okubo Toshimichi, a leading reformer during the Meiji Restoration. Harada noted that he was deeply interested in joining the film after witnessing the construction of Emperor Meiji's conference room on sound stage 19 (where Humphrey Bogart had once acted) at Warner Brothers studios.
- Shichinosuke Nakamura as Emperor Meiji. Credited with the implementation of the Meiji reforms to Japanese society, the Emperor is eager to import Western ideas and practices to modernize and empower Japan to become a strong nation. However, his inexperience causes him to rely heavily on the advice of men like Omura, who have their own agendas. His appearance bears a strong resemblance to Emperor Meiji during the 1860s (when his authority as Emperor was not yet firmly established) rather than during the 1870s, when the film takes place.
- Hiroyuki Sanada as Ujio, a master swordsman and one of Katsumoto's most trusted followers. Initially showing the most disdain towards Algren during the latter's time in captivity, he teaches Algren the art of sword fighting, coming to respect him as an equal. He is one of the last samurai to die in the final battle, being gunned down during Katsumoto's charge.
- Seizo Fukumoto as Silent Samurai, an elderly samurai tasked with monitoring Algren during his time in the village, who calls the samurai "Bob". "Bob" ultimately saves Algren's life (and speaking for the first and only time, "Algren-san!") by taking a bullet meant for him in the final battle.
- Billy Connolly as Sergeant Zebulon Gant, an Irish American Civil War veteran who served with and is loyal to Algren, persuading him to come to Japan and working with him to train the Imperial Army. During the first battle, he is killed by Hirotaro (Taka's husband) after being wounded with a spear.
- Shun Sugata as Nakao, a tall samurai who wields a naginata and is skilled in jujutsu. He assists Algren in rescuing Katsumoto and dies along with the other samurai in the final battle.
- Togo Igawa as General Hasegawa, a former Samurai serving in the Imperial Japanese Army. He commits seppuku after the first battle.
- John Koyama as Village Ninja
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