Cytoplasmic male sterility (CMS) serves as an effective strategy for harnessing hybrid vigor. NWB CMS in radish has found extensive application in hybrid seed production. The gene orf463a was recognized as a key candidate for causing NWB cytoplasmic male sterility in radish, however, the molecular basis of male sterility remain unclear. In this study, we revealed that accumulation of ORF463a in anthers correlates with microspore abortion in radish with NWB cytoplasm. Ectopic expression of orf463a with a mitochondrial transit peptide in Arabidopsis resulted in non-viable pollen and short siliques, which barely contained seeds, providing further verification of its function in inducing male sterility. Moreover, coIP and yeast two hybrid studies revealed that ORF463a interacted with the α subunit (ATP1), an essential part of mitochondrial complex V, which led to decreased complex V activity and ATP concentrations in NWB CMS lines during pollen development. To further elucidate the impact of energy deficiency on the biological processes involved in pollen development, a comparative transcriptome analysis was conducted, the results revealed that differentially expressed genes were predominantly associated with the asymmetric mitosis of mononuclear microspores, as well as the subsequent processes of pollen wall formation, anther dehiscence and pollen release. Collectively, a model was suggested for the mechanism of NWB CMS system. Our study demonstrated that orf463a is the causal gene for male sterility in NWB cytoplasm of radish. It interacts with ATP1, a component of complex V, impairing its enzyme activity, which disrupts mitochondrial energy production and ultimately causes pollen abortion.