Autophagy, a cellular survival mechanism, is thought to allow the recycling of cellular breakdown products when cancer cells are subjected to chemotherapy, thus decreasing drug-induced apoptosis. Disulfiram (DSF), a drug widely used to control alcoholism, possesses anticancer activity by inducing apoptosis in vitro and in vivo in a copper (Cu)-dependent manner. Our previous studies proved that DSF/Cu exerts increased anti-tumor effects on non-small cell lung cancer (NSCLC) xenograft models, and inhibits NSCLC recurrence driven by ALDH-positive cancer stem cells. The present study is designed to investigate whether DSF/Cu can induce autophagy in NSCLC cells and to determine the relationship between autophagy and apoptosis. First, we observed that DSF/Cu induced significant cytotoxicity and caspase-dependent apoptosis in NSCLC cells, accompanied by the formation of vacuoles in the cytoplasm. Next, we investigated the levels of autophagic markers, including LC3II (microtubule-associated protein 1 light chain 3), ATG5 and p62 with or without chloroquine by Western blot. In addition, we observed co-localization of LC3 and the lysosomal protein LAMP2 (lysosome-associated membrane protein 2) after treatment with DSF/Cu. The results showed that DSF/Cu induced autophagy. Finally, we demonstrated that DSF/Cu-induced apoptosis was greatly enhanced when autophagy was suppressed with Atg5 siRNA or 3-MA in NSCLC cells. This synergistic effect of DSF/Cu and 3-MA was further confirmed in the NSCLC xenograft model. Taken together, our results show that DSF/Cu stimulates autophagy in NSCLC cells, which is an impediment to DSF/Cu-induced apoptosis.