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.
| Home team | Result | Away team |
|---|---|---|
| Achilles '94 | 2–1 | Young FC Utrecht |
| Utrecht | 10–0 | BV Veendam |
| Emmen | 3–3 (p: 2-3) | TOP Oss |
| Go Ahead Eagles | 1–5 | Excelsior |
| Dordrecht '90 | 1–4 | Young PSV |
| SV TOP | 0–0 (p: 5-6) | VVV-Venlo |
| RKC Waalwijk E | 0–2 | Heracles |
| Sparta | 3–1 | Volendam (on September 23) |
| De Graafschap | 1–2 | MVV (on October 4) |
| Den Bosch | 3–2 | RBC (on October 18) |
| Home team | Result | Away team |
|---|---|---|
| ADO '20 | 1–2 | Cambuur |
| Willem II | 1–2 | FC Eindhoven |
| AZ | 3–2 | Rijnsburgse Boys |
| VV Noordwijk | 1–2 | NEC |
| SV Huizen | 1–4 | Groningen |
| Ter Leede | 1–2 (gg) | Fortuna Sittard |
| VV Baronie | 0–6 | NAC Breda |
| SV Urk | 2–3 (gg) | Helmond Sport |
| ADO Den Haag | 1–3 | FC Zwolle |
| Elinkwijk | 2–3 (gg) | Twente |
E one Eredivisie entrant
Second round
The matches of the second round were played between October 19, 2000 and January 23, 2001.
| Home team | Result | Away team |
|---|---|---|
| Fortuna Sittard | 4–0 | Groningen |
| FC Zwolle | 3–1 | NAC Breda |
| Helmond Sport | 1–3 | AZ |
| MVV | 0–3 | Utrecht |
| Young PSV | 0–1 | SBV Excelsior |
| Heracles | 3–2 | Cambuur |
| VVV-Venlo | 3–2 | TOP Oss |
| FC Eindhoven | 0–4 | Den Bosch |
| NEC | 3–1 | Sparta |
| Achilles '94 | 1–6 | Twente |
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.
| Home team | Result | Away team |
|---|---|---|
| PSV E | 4–1 | Den Bosch |
| NEC | 1–2 | Feyenoord E |
| Roda JC E | 2–0 | Utrecht |
| Excelsior | 1–3 | AZ |
| Ajax E | 1–2 (gg) | Vitesse E |
| Heracles | 1–5 | FC Zwolle |
| VVV-Venlo | 0–1 | Heerenveen E |
| Fortuna Sittard | 1–2 | Twente |
E six Eredivisie entrants
Quarter-finals
The first three matches were played on 7 February 2001. The fourth was played on 14 March.
| Home team | Result | Away team |
|---|---|---|
| FC Zwolle | 3–4 | Twente |
| Vitesse | 2–1 | AZ |
| Roda JC | 0–2 | PSV |
| Heerenveen | 4–3 | Feyenoord |
Semi-finals
The matches of the semi-finals were played on April 11 and 12, 2001.
| Home team | Result | Away team |
|---|---|---|
| Heerenveen | 1–3 | PSV |
| Vitesse | 0–0 (p: 3-4) | Twente |
Final
| PSV | 0–0 (a.e.t.) | Twente |
|---|---|---|
| Penalties | ||
Hofland  van Nistelrooy van Bommel de Jong  Waterreus Kolkka | 3 - 4 | Vennegoor of Hesselink Booth Heubach Karnebeek Polak Hulshoff |
Twente would participate in the UEFA Cup.