Cancer stem cells (CSCs) are considered the source of the initial tumor formation and postoperative recurrence and metastasis. CD133+ cells in hepatocellular carcinoma (HCC) display cancer stem-like properties and are thought to be responsible for chemoradioresistance. To explore the functional role of CD133 in liver cancer stem cells (LCSCs), we isolated CD133+ cells from the HCC cell line HepG2, which were tested and confirmed to be CSC-like cells in HCC, downregulated CD133 expression in HepG2-CD133+ cells by lentivirus-mediated short hairpin (shRNA) and analyzed the effects of CD133 on the modulation of stemness properties and chemoradiosensitivity in LCSCs. Our results showed that the in vitro cell proliferation, tumorsphere formation, colony formation and in vivo tumor growth in NOD/SCID mouse xenografts of LCSCs were significantly repressed after CD133 silencing. We also found that suppression of CD133 enhances the sensitivity of LCSCs to chemotherapy and radiotherapy. Knockdown of CD133 reduced G0/G1 phase cells and increased cellular apoptosis via modulation of Bcl-2 and Bax. Collectively, the stem-targeted therapy via CD133 could provide a novel strategy for the treatment of HCC.