The 1999–2000 KNVB Cup was the 82nd edition of the tournament, at the time known as the Amstel Cup for sponsorship reasons. The competition started on 31 July 1999 and the final was played on 21 May 2000. Roda JC beat NEC 2–0 and received the cup for the second time. A total of 86 clubs participated.
All 18 participants of the Eredivisie 1999–2000: six teams entered in the round of 16 of the knock-out stage; one team entered in the first round of the knock-out stage and the rest of the teams entered in the group stage.
The matches of the first round were played on September 22 and 23, 1999. sc Heerenveen entered the tournament this round, during the group stage they were still active in the Intertoto Cup.
The matches of the round of 16 were played on January 27, 28 and 30, 2000. The Eredivisie teams that had been playing in European competitions after qualification last season, entered the tournament this round.