Curcumin, a plant polyphenol, is a widely studied chemopreventive agent with demonstrated antitumor activities in preclinical studies and low toxicity profiles in multiple clinical trials against human malignancies. We previously showed that curcumin radiosensitizes cervical tumor cells without increasing the cytotoxic effects of radiation on normal human fibroblasts. Here we report that an inhibitory activity of curcumin on the antioxidant enzyme thioredoxin reductase-1 (TxnRd1) is required for curcumin-mediated radiosensitization of squamous carcinoma cells. Stable knockdown of TxnRd1 in both HeLa and FaDu cells nearly abolished curcumin-mediated radiosensitization. TxnRd1 knockdown cells showed decreased radiation-induced reactive oxygen species and sustained extracellular signal-regulated kinase 1/2 activation, which we previously showed was required for curcumin-mediated radiosensitization. Conversely, overexpressing catalytically active TxnRd1 in HEK293 cells, with low basal levels of TxnRd1, increased their sensitivity to curcumin alone and to the combination of curcumin and ionizing radiation. These results show the critical role of TxnRd1 in curcumin-mediated radiosensitization and suggest that TxnRd1 levels in tumors could have clinical value as a predictor of response to curcumin and radiotherapy.