Alki Larnaca won their 3rd title.
Fourteen teams participated in the 2000–01 Cypriot Second Division.
The team with the most points at the end of the season crowned champions.
The first three teams were promoted to 2001–02 Cypriot First Division and the last three teams were relegated to the 2001–02 Cypriot Third Division.
Teams promoted to 2000–01 Cypriot First Division Teams relegated from 1999–2000 Cypriot First Division Teams promoted from 1999–2000 Cypriot Third Division Teams relegated to 2000–01 Cypriot Third Division