Verticillium wilt, caused by the fungus Verticillium dahliae, is a major plant disease leading to severe global agricultural losses. While the interaction between V. dahliae and plants has been widely studied, how metabolism regulation influences fungal development and pathogenicity remains unclear. Fatty acids are key metabolites and utilized for S-acylation to modulates the properties and functions of proteins, yet this post-translational lipidation has not been reported in V. dahliae. Here, systematic analysis showed that the protein S-acyltransferase VdERF2 is essential for the development of V. dahliae and its pathogenicity toward host plants. Comparative S-acyl-proteomic analyses revealed that VdERF2-dependent S-acylation is highly enriched in metabolic processes, especially purine metabolism. Among the identified substrates, we found VdHIUH, an enzyme that converts uric acid to allantoin in purine metabolism, whose S-acylation is critical for fungal development and virulence. VdERF2-mediated S-acylation enhances the activity of VdHIUH, thereby reducing uric acid accumulation. Allantoin alleviates the increase of reactive oxygen species caused by defective VdHIUH S-acylation, restoring fungal development. In conclusion, this study uncovers a crucial role of S-acylation in regulating purine metabolism in V. dahliae, providing new insights into the molecular basis of fungal pathogenicity and potential strategies for improving crop resistance to Verticillium wilt.