Heat shock protein 60 (Hsp60), abundantly presents in mitochondria, is a highly conserved chaperone that maintains the stability and functionality of mitochondrial proteins, while also participating in the regulation of various cellular processes. As a member of the heat shock family, Hsp60 significantly influences viral proliferation. However, limited research is available on its role in the proliferation of entomopathogenic baculoviruses, particularly Bombyx mori nucleopolyhedrovirus (BmNPV). Our previous proteomics results showed a significant decrease of Hsp60 acetylation levels after BmNPV infection. To investigate the impact of Hsp60 deacetylation on viral proliferation, site-direct mutagenesis was performed to generate a deacetylated (K/R) mimic of Hsp60. We found that the acetylation level of lysine 362 (K362) decreased after BmNPV challenge. Furthermore, overexpression of deacetylation-mimicking Hsp60 reduced the chaperone activity of Hsp60, leading to impaired mitochondrial function, including increased reactive oxygen species (ROS) levels, decreased mitochondrial membrane potential, and reduced substrate protein Manganese-containing superoxide dismutase (Mn-SOD) activities, ultimately leading to inhibition of viral proliferation. This study establishes lysine 362 acetylation of Hsp60 as a model for Posttranslational modifications induced by host-virus interactions, providing new insights into potential antiviral strategies.