New radiosensitizers are urgently needed for radiation therapy patients with localized hepatocellular carcinoma (HCC) that is refractory to radical surgery. We previously found that genistein, a major soy isoflavone, exerts radioprotective effects on L-02 normal liver cells at low concentrations. Here, we report that 5 µM genistein shows less harm to L-02 cells than HCC cells and that it significantly enhances the radiosensitivity of HCC cells by enhancing DNA damage, chromosomal aberrations and cell cycle arrest at G₂/M phase and by exacerbating apoptosis. Mechanistically, genistein aggravates X-ray-induced decreases in the levels of phospho-Bad (Ser136) but enhances the levels of phospho-Chk2 (Thr68), phospho-ATM (Ser1981) and γ-H2AX. Micro-array analysis indicated that downregulation of POU6F and CCNE2 expression and upregulation of FBXO32 and cyclin B1 expression might play vital roles in genistein-induced radiosensitivity. These findings suggest genistein as an interesting candidate for adjuvant radiotherapy for HCC and indicate that genistein causes less harm to normal cells than HCC cells by inducing G₂/M arrest and apoptosis.