The first round of the 2000–01 UEFA Cup began on 14 September 2000.
The round included 41 winners from the qualifying round, 16 losing teams from the Champions League 3rd qualifying round, 3 winners of the Intertoto Cup and 36 new entrants.
[2] Werder Bremen won 6–2 on aggregate.
3–3 on aggregate; VfB Stuttgart won on away goals.
Lokomotiv Moscow won 4–2 on aggregate.
Celta Vigo won 1–0 on aggregate.
Red Star Belgrade won 4–2 on aggregate.
2–2 on aggregate; MTK Budapest won on away goals.
5–5 on aggregate; Wisła Kraków won 4–3 on penalties.
Rayo Vallecano won 2–1 on aggregate.