Targeting hypoxia-inducible factor-1 (HIF-1) active cells in tumors may be an excellent strategy to improve the outcome of radiation therapy. On the basis of the reported role of YC-1 as a HIF-1 inhibitor with anti-cancer activity, we tested the therapeutic efficacy of YC-1 against radioresistance in vitro. The AMC-HN3 cancer cell line, developed from squamous cell carcinoma of the larynx, was cultured under hypoxic conditions or in the presence of cobalt chloride. Both treatments induced nuclear accumulation of HIF-1alpha protein. Cells cultured under normoxic or hypoxic conditions with and without YC-1 treatment were irradiated and analyzed using flow cytometry and clonogenic assays. In the absence of YC-1 treatment, irradiation induced a greater cytotoxic effect in normoxic cells than in cobalt-treated cells. Treatment of cobalt-treated cells with YC-1 effectively inhibited HIF-1alpha expression, and enhanced the sensitivity of cells to radiation, decreasing the surviving fraction to that of normoxic cells. Flow cytometry confirmed these results, showing that the sub-G1 fraction was increased in YC-1-treated hypoxic cells after irradiation. Our results suggest that YC-1 treatment may be an effective therapeutic strategy for overcoming the radioresistance of HIF-1alpha-expressing, hypoxic cancer cells.