| Competition: | Cypriot Second Division |
| Season: | 2000–01 |
| Winners: | Alki (3rd title) |
| Matches: | 182 |
| Total Goals: | 591 |
| Prevseason: | 1999–00 |
| Nextseason: | 2001–02 |
The 2000–01 Cypriot Second Division was the 46th season of the Cypriot second-level football league. Alki Larnaca won their 3rd title.
Fourteen teams participated in the 2000–01 Cypriot Second Division. All teams played against each other twice, once at their home and once away. 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