BACKGROUND & AIMS: Systemic chemotherapy serves as an adjuvant treatment for post-operation patients with hepatocellular carcinoma (HCC), and provides curative option for the patients with unresectable HCC. However, its efficiency is largely limited because of the high incidence of chemo-resistance. Increasing evidence has shown that tumor initiating cells (TICs) not only have the ability to self-renew and drive the initiation and progression of cancer, but also exhibit greater resistance to conventional chemo- and radio-therapies than non-TICs. It was the aim of this study to investigate the effects of ATRA with and without cisplatin on TIC differentiation and apoptosis in human HCC. METHODS: In the present study, we evaluated the TICs of HCC cell differentiation induced by all-trans retinoic acid (ATRA), and developed a novel chemotherapeutic approach to HCC, by characterizing the function of combinatorial treatment with cis-diammineplatinum(II) (cisplatin) and ATRA in vitro and in vivo. RESULTS: ATRA effectively induced differentiation of TICs, which potentiated the cytotoxic effects of cisplatin. The combinatorial treatment of ATRA acid and cisplatin reduced protein kinase B (AKT) (Thr308) phosphorylation, and promoted apoptosis of HCC cells more significantly than treatment with cisplatin alone. In addition, the combined treatment with the two drugs exerted stronger inhibition on either HCC cell migration in vitro or metastasis in vivo, when compared to the treatment with either drug alone. CONCLUSIONS: These results indicated that ATRA could significantly improve the effect of cisplatin, which is at least partially attributed to ATRA-induced differentiation of HCC TICs, and the subsequent decrease in this chemo-resistant subpopulation.