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BACKGROUND At present, the treatment of hepatocellular carcinoma (HCC) is disturbed by the treatment failure and recurrence caused by the residual liver cancer stem cells (CSCs). Therefore, drugs targeting HCC CSCs should be able to effectively eliminate HCC and prevent its recurrence. In this study, we demonstrated the effect of Polyphyllin VII (PP7) to HCC CSCs, and explored their potential mechanism. METHODS HepG2 and Huh7 cells, were used to analyze the antitumor activity of PP7 by quantifying cell growth and metastasis as well as to study the effect on stemness. RESULTS Our results demonstrated that PP7 promoted apoptosis and significantly inhibited proliferation and migration of both HepG2 and Huh7 cells. PP7 also inhibited tumor spheroid formation and induced significant changes in the expression of stemness markers (CD133 and OCT-4). These effects of PP7 were mediated by the STAT3 signaling. CONCLUSION PP7 can effectively suppress tumor initiation, growth, metastasis, and inhibit stemness through regulation of STAT3 signaling pathway in liver cancer cells. Our data would add more evidence to further clarify the therapeutic effect of PP7 against HCC.