The 2000–01 season was the 86th season of the Isthmian League, which is an English football competition featuring semi-professional and amateur clubs from London, East and South East England.
Slough Town, Carshalton Athletic and Dulwich Hamlet finished bottom of the table and relegated to Division One.
Three fixtures left unfulfilled due to bad weather conditions.
Division Three consisted of 22 clubs, including 17 clubs from the previous season and five new teams: Arlesey Town and Ashford Town both debuted in the league and achieved a promotion along with Lewes.
Two fixtures left unfulfilled due to bad weather conditions.