Lysine acetylation is one of the most prevalent post-translational modifications and has since been found to regulate histone functions and affect chromatin structure and gene expression. Here, we charactered Rpd3, a histone deacetylase of Beauveria bassiana, played important roles in the deacetylation of histone H3 and H4. We unveil the role of Rpd3 in asexual development and show that a ΔRpd3 mutant strain has severe growth defects and is defective in conidiophores and blastospores development. Proteomic analyses indicated that Rpd3 functions in asexual development and virulence and multiple stress responses via broad genetic pathways in B. bassiana. Therefore, we conclude that BbRpd3 is vital for asexual development, virulence and multiple stress responses in Beauveria bassiana.