Williams syndrome transcription factor (WSTF) is a transcription factor and tyrosine kinase. WSTF overexpression promotes migration and proliferation of various cancers, and Ser158 (WSTFS158) phosphorylation plays an important role in this process. However, the role of the other posttranslational modifications of WSTF is unknown. Here, we report that lysine (K) 426 on WSTF is acetylated by MOF and deacetylated by SIRT1. Mechanistically, male-specific lethal (MSL) 1v1 interaction with WSTF facilitates its interaction with MOF for WSTF acetylation, which in turn promotes WSTFS158 phosphorylation. The kinase and transcriptional regulatory activity of WSTF were enhanced by acetylation. WSTFK426ac levels positively and significantly correlated with tumor size, histological grade, and age. Moreover, we demonstrated that acetylated WSTF promotes cancer cell proliferation, migration, invasion, and tumor formation. In conclusion, we identified the enzymes regulating WSTF K426 acetylation, and demonstrated an acetylation-dependent mechanism that modulates the activities of WSTF and contributes to tumorigenesis. Our findings provide new clues to study WSTF-mediated normal development and disease.