We investigated the effects of mitochondrial damage via carbon ion irradiation on the proliferation of human non-small cell lung cancer (A549) cells by monitoring mitochondrial morphological changes, mitochondrial membrane potential transformation, release of membrane-associated proteins, and mitophagy. A549 cell proliferation and activity were monitored by CCK-8 and colony formation assays. Changes in mitochondrial morphology and mitochondrial membrane potential were detected by mitochondrial membrane-specific fluorescence staining. Expression of associated proteins was detected by western blot and real-time quantitative PCR. Annexin V-FITC and PI staining were used to detect cell apoptosis. Mitophagy was detected by immunofluorescence co-localization and western blot. The apoptosis rate of mitochondria-lacking A549 cells after carbon ion irradiation was also quantitated. Irradiation with carbon ions at dose of 4 Gy significantly depressed the proliferation capacity of A549 cells, and induced mitochondrial shrinkage and decreased mitochondrial membrane potential. Levels of mitochondrial membrane protein Bax/Bak, and intermembrane proteins Cyto-C and SMAC were elevated, with increased apoptosis and mitophagy. In cells depleted of mitochondria, carbon ion irradiation exerted no significant effect on A549 cell apoptosis. Carbon ion irradiation can inhibit A549 cell proliferation. The half maximal inhibitory exposure dose is 4 Gy. Carbon ions lose their anti-proliferative function in mitochondria-free A549 cells. In summary, carbon ion irradiation can decrease mitochondrial membrane potential and induce the release of pro-apoptotic proteins, thereby inhibiting A549 cell proliferation.