Genistein (GEN) has been previously reported to enhance the radiosensitivity of cancer cells; however, the detailed mechanisms remain unclear. Here, we report that GEN treatment inhibits the cytoplasmic distribution of Bcl-xL and increases nuclear Bcl-xL in non-small cell lung cancer (NSCLC). Interestingly, our in vitro data show that ionizing radiation IR treatment significantly increases IR-induced DNA damage and apoptosis in a low cytoplasmic Bcl-xL NSCLC cell line compared to that of high cytoplasmic Bcl-xL cell lines. In addition, clinical data also show that the level of cytoplasmic Bcl-xL was negatively associated with radiosensitivity in NSCLC. Furthermore, we demonstrated that GEN treatment enhanced the radiosensitivity of NSCLC cells partially due to increases in Beclin-1-mediated autophagy by promoting the dissociation of Bcl-xL and Beclin-1. Taken together, these findings suggest that GEN can significantly enhance radiosensitivity by increasing apoptosis and autophagy due to inhibition of cytoplasmic Bcl-xL distribution and the interaction of Bcl-xL and Beclin-1 in NSCLC cells, respectively.