Recent studies have implicated autophagy in numerous cellular responses to stress. During the establishment of human lung cancer cell lines without mitochondrial DNA, a significant depopulation of mitochondria occurred that was accompanied by the loss of the mitochondrial membrane potential. Notably, we observed autophagy in ethidium bromide (EtBr)-induced mitochondrial degradation. In the present study, we confirmed the involvement of autophagy in mitochondrial degradation after exposure to a low concentration of EtBr. Lung cancer cells undergoing mitochondrial autophagy exhibited a slower growth rate in vitro and in vivo. Furthermore, the degradation was mediated by the class III phosphatidylinositol 3-kinase (PI3K)-Beclin-1 complex. These findings indicate that autophagy is responsible for EtBr‑induced mitochondrial degradation via the PI3K‑Beclin-1 signaling pathway.