Cytoplasmic male sterility (CMS) is a powerful tool for the exploitation of hybrid heterosis and the study of signaling and interactions between the nucleus and the cytoplasm. C-type CMS (CMS-C) in maize has long been used in hybrid seed production, but the underlying sterility  factor and its mechanism of action remain unclear. In this study, we demonstrate that the mitochondrial gene atp6c confers male sterility in CMS-C maize. The ATP6C protein showed stronger interactions with ATP8 and ATP9 than ATP6 in the assembly of F1Fo-ATP synthase (F-type ATP synthase, ATPase), thereby reducing the quantity and activity of assembled F1Fo-ATP synthase. By contrast, the quantity and activity of the F1' component were increased in CMS-C lines. Reduced F1Fo-ATP synthase activity caused accumulation of excess protons in the inner membrane space of  the mitochondria, triggering a burst of reactive oxygen species (ROS), premature programmed cell death of the tapetal cells, and pollen abortion. These findings locate the sterility gene, document the contribution of ATP6C to F1Fo-ATP synthase assembly, and provide novel insights into the molecular mechanisms of male sterility in CMS-C maize.