Fusarium proliferatum is the causative agent of rice spikelet rot disease, which can produce a group of toxic secondary metabolites, especially fumonisins. Velvet complex is a master regulator governing the development processes and secondary metabolism in filamentous fungi. In this study, we investigated the biological functions of velvet members FpVeA and FpVelB in F. proliferatum. Compared with the wild-type Fp9 strain, deletion of FpveA or FpvelB genes resulted in retarded hyphal growth but promoted conidiation. Disruption mutants exhibited decreased conidial trehalose contents and enhanced sensitivity to H2O2 stress, as well as inducing expression of photoreceptors. Notably, inactivation of FpveA or FpvelB led to a reduction in production of fumonisin B1 (FB1), coinciding with downregulation of fumonisin biosynthetic genes. Furthermore, the absence of FpveA or FpvelB displayed attenuated virulence toward rice spikelets, accompanied by fewer invasive hyphae and a failure of penetration ability. Taken together, these results demonstrated that FpVeA and FpVelB play crucial roles in the asexual development, oxidative stress, toxin synthesis and pathogenicity of F. proliferatum.