This round marked the first time First Division and Premier League (top-flight) teams played.
The biggest surprises of the round saw Tranmere Rovers complete one of the greatest FA Cup comebacks ever when they beat Southampton 4–3 in a replay after they had been trailing 3–0 at half time.
(with 36-year-old former Southampton striker Paul Rideout scoring a hat-trick for Tranmere), while Wycombe Wanderers reached the quarter-finals for the first time in their history with a penalty shoot-out win over Wimbledon that follow two 2–2 draws.
Unlike earlier rounds, matches were played on neutral grounds on Sunday, 8 April 2001.
The third round replay between Dagenham & Redbridge vs. Charlton Athletic was originally scheduled to be on Sky Sports on 17th January 2001, but because of a frozen pitch, the game was postponed.