Autophagy is an evolutionarily conserved cellular response to conditions of stress such as hypoxia, which induce radioresistance in cancer cells. We studied the mechanism of action of hypoxia on autophagy and radiosensitivity in colon cancer cells. In the human colon cancer cell lines SW480 and SW620, autophagosomes were analyzed to evaluate autophagy by flow cytometry. The expression of hypoxia inducible factor-1α (HIF-1α), Bcl-2, and miR-210 was detected by western blotting and quantitative real-time polymerase chain reaction (PCR). HIF-1α and miR-210 inhibition was induced by siRNA transfections. Apoptosis detection and colony assays were performed to determine radiosensitivity. HIF-1α and miR-210 showed a significant increase under hypoxic condition. The inhibition of HIF-1α decreased miR-210 expression and autophagy. Silencing of miR-210 upregulated Bcl-2 expression and reduced the survival fraction of colon cancer cells after radiation treatment. Under hypoxia, HIF-1α induces miRNA-210 which in turn enhances autophagy and reduces radiosensitivity by downregulating Bcl-2 expression in colon cancer cells. Our results imply that autophagy contributes to the reduction of radiosensitivity in hypoxic environment, and the process is mediated through the HIF-1α/miR-210/Bcl-2 pathway in human colon cancer cells.