| Year: | 2000–01 |
| Country: | Cyprus |
| Num Teams: | 50 |
| Dates: | 11 November 2000 – 12 May 2001 |
| Champions: | Apollon (5th title) |
| Nextseason: | 2001–02 |
The 2000–01 Cypriot Cup was the 59th edition of the Cypriot Cup. A total of 50 clubs entered the competition. It began on 11 November 2000 with the preliminary round and concluded on 12 May 2001 with the final which was held at GSP Stadium. Apollon Limassol won their 5th Cypriot Cup trophy after beating Nea Salamina 1–0 in the final.
In the 2000–01 Cypriot Cup, participated all the teams of the Cypriot First Division, the Cypriot Second Division, the Cypriot Third Division and 8 of the 14 teams of the Cypriot Fourth Division.
The competition consisted of six knock-out rounds. In the preliminary round and in the first round each tie was played as a single leg and was held at the home ground of one of the two teams, according to the draw results. Each tie winner was qualifying to the next round. If a match was drawn, extra time was following. If extra time was drawn, there was a replay at the ground of the team who were away for the first game. If the rematch was also drawn, then extra time was following and if the match remained drawn after extra time the winner was decided by penalty shoot-out.
The next three rounds were played in a two-legged format, each team playing a home and an away match against their opponent. The team which scored more goals on aggregate, was qualifying to the next round. If the two teams scored the same number of goals on aggregate, then the team which scored more goals away from home was advancing to the next round. If both teams had scored the same number of home and away goals, then extra time was following after the end of the second leg match. If during the extra thirty minutes both teams had managed to score, but they had scored the same number of goals, then the team who scored the away goals was advancing to the next round (i.e. the team which was playing away). If there weren't scored any goals during extra time, the qualifying team was determined by penalty shoot-out.
The cup winner secured a place in the 2001–02 UEFA Cup.
All the 14 clubs of the Cypriot Second Division, all the 14 clubs of the Cypriot Third Division and 8 clubs from the Cypriot Fourth Division (first eight of the league table the day of draw) participated in the preliminary round.
The 14 clubs of the Cypriot First Division advanced directly to the first round and met the winners of the preliminary round ties: