Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for the anti-tumor effects, including the inhibition of tumor cell growth, metastasis and angiogenesis. In this study, we investigated the effect of Wit A on radiation-induced apoptosis in human renal cancer cells (Caki cells). Our results showed that, compared with Wit A or radiation alone, the combination of both resulted in a significant enhancement of apoptosis, showing the increase in the cleavage of caspase-3 and PARP as well as sub-G1 cell population. In addition, reactive oxygen species (ROS) generation was correlated with the enhancement of radiation-induced apoptosis by Wit A. Wit A downregulated Bcl-2 protein levels and ectopic expression of Bcl-2 in Caki cells attenuated the apoptosis induced by Wit A plus radiation. Taken together, these results indicate that Wit A enhanced radiation-induced apoptosis in Caki cells through ROS generation, down-regulation of Bcl-2 and Akt dephosphorylation. Thus, our study shows that Wit A may be used as an effective radiosensitizer in cancer therapy.