The land of Lord Gohda is in unrest; rumors of corruption and betrayal are whispered among his subjects. Gohda orders his two top ninja, Rikimaru and Ayame, to investigate the rumors. At the same time, Gohda's retainer Sekiya arranges a meeting with a fortune-teller, but the fortune-teller turns out to be an agent of evil, kidnapping Gohda's only daughter, Kiku. Ayame runs off in a blind rage to rescue Kiku, while Rikimaru continues to investigate the strange rumors spreading across the land.

Tenchu: Shadow Assassins is a third-person stealth action game and part of the long-running Tenchu franchise. The main story is broken into 10 missions, with the player taking control of either Rikimaru or Ayame as the story dictates. The game has a much stronger focus on stealth than previous titles. Enemies can be engaged in open combat only if the player is armed with a sword; if not, they must use ninjutsu to escape and retreat to the last checkpoint. Each mission has a number of objectives, which must be completed to clear the stage and move on. In addition to the story mode, there are also 50 "assignments", which are shorter missions that often focus on a specific technique.