The PI3K/AKT/mTOR pathway and autophagy are known to play important roles in cancer radioresistance. The aim of the present study was to investigate whether the combination of temsirolimus (TEM), an mTOR inhibitor, and chloroquine (CQ), an autophagy inhibitor, can increase radiosensitivity in colorectal cancer (CRC) cells. The efficacies of TEM and/or CQ as radiosensitizers were examined using clonogenic assays in CRC cell lines SW480 and HT‑29. The expression levels of the phosphorylated isoforms of S6 and 4E‑BP1, downstream proteins of mTOR, as well as the expression levels of p62 and LC3, autophagy‑related proteins, were assessed by western blot analysis. The formation of acidic organelles was detected in acridine orange‑stained cells. Apoptosis and caspase activity were assessed using flow cytometry. The results revealed that ionizing radiation (IR) activated the downstream proteins of mTOR and induced autophagy. In the clonogenic assays, neither TEM nor CQ influenced the efficacy of IR, whereas their combination significantly increased the dose‑dependent efficacy of IR. TEM inhibited phosphorylation of the downstream proteins of mTOR and induced autophagy. CQ inhibited autophagy in the late phase and did not influence the downstream proteins of mTOR. TEM and CQ inhibited both the phosphorylation of downstream proteins of mTOR and autophagy. Cell death analysis revealed that the combination of TEM and CQ strongly induced apoptosis in cells exposed to IR. In conclusion, the combination of TEM and CQ increased radiosensitivity in CRC cells through co‑inhibition of mTOR and autophagy.