The 2000-2001 KNVB Cup (at the time called Amstel Cup) was the 83rd edition of the Dutch national football annual knockout tournament for the KNVB Cup. 86 teams contested, beginning on 8 August 2000 and ending at the final on 24 May 2001. Roda JC unsuccessfully defended its 2000 title in the Quarter-finals losing to PSV, 0–2. Twente successfully pursued its 2nd #Final KNVB Cup on 24 May 2001 in De Kuip, Rotterdam losing to PSV, 0–0 (penalty score, 3-4). 45,000 attended. Twente contested the UEFA Cup.

Teams

Group stage

The matches of the group stage were played between August 8 and September 13, 2000. 79 teams participated and 117 matches were played. 39 teams advanced to the next round. E Eredivisie; 1 Eerste Divisie; A Amateur teams

Knock-out Stage

First round

The matches of the first round were played on September 20 and 21, 2000. RKC Waalwijk entered the tournament here, during the group stage they were still active in the Intertoto Cup. E one Eredivisie entrant

Second round

The matches of the second round were played between October 19, 2000 and January 23, 2001.

Round of 16

The matches were played between January 24 and 27, 2001. The six Eredivisie teams that had been playing in European competitions after qualification last season, entered the tournament this round. E six Eredivisie entrants

Quarter-finals

The first three matches were played on 7 February 2001. The fourth was played on 14 March.

Semi-finals

The matches of the semi-finals were played on April 11 and 12, 2001.

Final

Twente would participate in the UEFA Cup.