The 2000–01 Czech Cup was the eighth edition of the annual football knockout tournament organized by the Czech Football Association of the Czech Republic.
26 teams took part in the preliminary round.
83 teams entered the competition at this stage.
The fourth round was played between 20 and 25 March 2001.
The final was played with the golden goal rule; in the event of a tie after 90 minutes, 30 minutes additional time would be added, with any goal signalling the immediate end of the game.