Fusarium wilt is one of the most serious diseases affecting cotton. However, the pathogenesis and mechanism by which F. oxysporum overcomes plant defence responses are unclear. Here, a new group D MAPK gene, GhMPK20, was identified and functionally analysed in cotton. GhMPK20 expression was significantly induced by F. oxysporum. Virus-induced gene silencing (VIGS) of GhMPK20 in cotton increased the tolerance to F. oxysporum, while ectopic GhMPK20 overexpression in N. benthamiana reduced F. oxysporum resistance via disrupting the SA-mediated defence pathway. More importantly, an F. oxysporum-induced MAPK cascade pathway composed of GhMKK4, GhMPK20 and GhWRKY40 was identified. VIGS of GhMKK4 and GhWRKY40 also enhanced F. oxysporum resistance in cotton, and the function of GhMKK4-GhMPK20 was shown to be essential for F. oxysporum-induced GhWRKY40 expression. Together, our results indicated that the GhMKK4-GhMPK20-GhWRKY40 cascade in cotton plays important roles in the pathogenesis of F. oxysporum. This research broadens our knowledge of the negative role of the MAPK cascade in disease resistance in cotton and provides an important scientific basis for formulating Fusarium wilt prevention strategies. This article is protected by copyright. All rights reserved.