Protein palmitoylation, one of posttranslational modifications, is catalyzed by a group of palmitoyl transferases (PATs) and plays critical roles in the regulation of protein functions. However, little is known about the function and mechanism of PATs in plant pathogenic fungi. The present study reports the function and molecular mechanism of FonPATs in Fusarium oxysporum f. sp. (), the causal agent of watermelon Fusarium wilt. The genome contains six genes with distinct functions in vegetative growth, conidiation and conidial morphology, and stress response. FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for virulence on watermelon mainly through regulating fungal growth within host plants. Comparative proteomics analysis identified a set of proteins that were palmitoylated by FonPAT2, and some of them are previously reported pathogenicity-related proteins in fungi. The FonAP-2 complex core subunits FonAP-2α, FonAP-2β, and FonAP-2μ were palmitoylated by FonPAT2 in . FonPAT2-catalyzed palmitoylation contributed to the stability and interaction ability of the core subunits to ensure the formation of the FonAP-2 complex, which is essential for vegetative growth, asexual reproduction, cell wall integrity, and virulence in . These findings demonstrate that FonPAT1, FonPAT2, and FonPAT4 play important roles in virulence and that FonPAT2-catalyzed palmitoylation of the FonAP-2 complex is critical to virulence, providing novel insights into the importance of protein palmitoylation in the virulence of plant fungal pathogens. Fusarium oxysporum f. sp. (), the causal agent of watermelon Fusarium wilt, is one of the most serious threats for the sustainable development of the watermelon industry worldwide. However, little is known about the underlying molecular mechanism of pathogenicity in . Here, we found that the palmitoyl transferase () genes play distinct and diverse roles in basic biological processes and stress response and demonstrated that FonPAT1, FonPAT2, and FonPAT4 have PAT activity and are required for virulence in . We also found that FonPAT2 palmitoylates the core subunits of the FonAP-2 complex to maintain the stability and the formation of the FonAP-2 complex, which is essential for basic biological processes, stress response, and virulence in . Our study provides new insights into the understanding of the molecular mechanism involved in virulence and will be helpful in the development of novel strategies for disease management.