Aims Mitochondrial fission is an essential dynamics that maintains mitochondrial morphology and function. This study seeks to determine the roles of mitochondrial fission in the filamentous entomopathogenic fungusBeauveria bassiana. Material and methods Three fission-related genes (BbFis1,BbMdv1andBbDnm1) were functionally characterized via protein intracellular localization and construction of gene disruption mutants. Results Mitochondrial localization was only observed for BbFis1 which interacts with BbMdv1, but BbMdv1 did not have interaction with BbDnm1. Single disruption mutant of three genes generated the elongated and enlarged mitochondria which could not be eliminated via the mitophagy. Three mutant strains displayed the reduced ATP synthesis and vegetative growth compared with the wild type. Three genes were involved in the early stage of conidiation and unnecessary for the late stage. However, all three genes significantly contribute to blastospore development under submerged condition, and the loss ofBbMdv1had the greatest effects compared with the losses ofBbFis1orBbDnm1. Finally, disruption of three genes significantly attenuated fungal virulence, but their mutations had different influences. Conclusions In addition to their consistent roles in mitochondrial division and mitophagy, three fission-related genes perform divergent roles in the development and virulence of the entomopathogenic fungusB. bassiana. Significance and Impact of the Study This study shows that mitochondrial fission is associated with lifecycle ofB. bassiana. These findings provide information for the manipulation of fungal physiology and facilitate the application of entomopathogenic fungi.