Fusarium oxysporum f. sp. (), a soilborne phytopathogenic fungus, causes watermelon Fusarium wilt, resulting in serious yield losses worldwide. However, the underlying molecular mechanism of virulence is largely unknown. The present study investigated the biological functions of six , encoding RNA binding Pumilio proteins, and especially explored the molecular mechanism of in virulence. A series of phenotypic analyses indicated that have distinct but diverse functions in vegetative growth, asexual reproduction, macroconidia morphology, spore germination, cell wall, or abiotic stress response of . Notably, the deletion of attenuates virulence by impairing the invasive growth and colonization ability inside the watermelon plants. FonPUF1 possesses RNA binding activity, and its biochemical activity and virulence function depend on the RNA recognition motif or Pumilio domains. FonPUF1 associates with the actin-related protein 2/3 (ARP2/3) complex by interacting with FonARC18, which is also required for virulence and plays an important role in regulating mitochondrial functions, such as ATP generation and reactive oxygen species production. Transcriptomic profiling of identified a set of putative FonPUF1-dependent virulence-related genes in , possessing a novel A-rich binding motif in the 3' untranslated region (UTR), indicating that FonPUF1 participates in additional mechanisms critical for virulence. These findings highlight the functions and molecular mechanism of FonPUFs in virulence. Fusarium oxysporum is a devastating plant-pathogenic fungus that causes vascular wilt disease in many economically important crops, including watermelon, worldwide. F. oxysporum f. sp. () causes serious yield loss in watermelon production. However, the molecular mechanism of Fusarium wilt development by remains largely unknown. Here, we demonstrate that six putative Pumilio proteins-encoding genes () differentially operate diverse basic biological processes, including stress response, and that is required for virulence. Notably, FonPUF1 possesses RNA binding activity and associates with the actin-related protein 2/3 complex to control mitochondrial functions. Furthermore, FonPUF1 coordinates the expression of a set of putative virulence-related genes in by binding to a novel A-rich motif present in the 3' UTR of a diverse set of target mRNAs. Our study disentangles the previously unexplored molecular mechanism involved in regulating virulence, providing a possibility for the development of novel strategies for disease management.