Gozo and Żurrieq were the relegated teams from the 1999–2000 Maltese Premier League.
Marsaxlokk and Qormi were the promoted teams from the Maltese Second Division.
Marsa finished as champions, by just one point, and were promoted to the 2001–02 Maltese Premier League alongside Lija Athletic, who finished runners-up.
Tarxien Rainbows and Żurrieq were relegated to the 2001–02 Maltese Second Division.
The Maltese First Division 2000–01 was made up of these teams: For a complete set of results, see 1.