In a variety of cancer cell types, the pharmacological and genetic blockade of autophagy increases apoptosis induced by various anticancer drugs. These observations suggest that autophagy counteracts drug‑induced apoptosis. We previously reported that in human melanoma and osteosarcoma cells, autophagy inhibitors, such as 3‑methyladenine and chloroquine increased the sensitivity to apoptosis induced by tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL). In the present study, we report that different autophagy inhibitors regulate the mitochondrial network and calcium (Ca2+) dynamics in these cells. We found that compared to tumor cells, normal fibroblasts were more resistant to the cytotoxicity of TRAIL and autophagy inhibitors used either alone or in combination. Notably, TRAIL increased the autophagic flux in the tumor cells, but not in the fibroblasts. Live‑cell imaging revealed that in tumor cells, TRAIL evoked modest mitochondrial fragmentation, while subtoxic concentrations of the autophagy inhibitors led to mitochondrial fusion. Co‑treatment with TRAIL and subtoxic concentrations of the autophagy inhibitors resulted in severe mitochondrial fragmentation, swelling and clustering, similar to what was observed with autophagy inhibitors at toxic concentrations. The enhanced aberration of the mitochondrial network was preceded by a reduction in mitochondrial Ca2+ loading and store‑operated Ca2+ entry. On the whole, the findings of this study indicate that co‑treatment with TRAIL and autophagy inhibitors leads to increased mitochondrial Ca2+ and network dysfunction in a tumor‑selective manner. Therefore, the co‑administration of TRAIL and autophagy inhibitors may prove to be a promising tumor‑targeting approach for the treatment of TRAIL‑resistant cancer cells.