Objective: To investigate the influence of cucurbitacin I on growth of hepatocellular carcinoma (HCC) cells and the mechanism. Methods: The influences of cucurbitacin I on proliferative activities of different types of HCC cells (HepG2, QGY-7703 and SMMC-7721) were measured by CCK-8 assay. In HepG2 cells after treatment with cucurbitacin I, the colony formation, cell cycle profile and apoptosis were examined by plate colony-forming assay, flow cytometry and Hochest 33342 staining, and the protein and mRNA expressions of anti-apoptotic factors and their related signaling molecules were determined by Western blot analysis and qRT-PCR method, respectively. Results: The proliferations of all the selected HCC cells were significantly inhibited by cucurbitacin I treatment, with a time- and concentration-dependent manner (all P<0.05), and the 48-h IC50 value of cucurbitacin I for HepG2, QGY-7703 and SMMC-7721 was 0.19, 4.16 and 1.13 μmol/L, respectively. In HepG2 cells after cucurbitacin I treatment for 24 h, the clone formation was almost completely suppressed, typical apoptotic morphological changes and evident G2-phase arrest were presented, and both protein and mRNA expressions of the anti-apoptotic factors Mcl-1 and survivin as well as the transcription factor STAT3 were markedly down-regulated, and the semi-quantitative analysis of mRNA expressions showed all differences had statistical significance (all P<0.05). Conclusion: Cucurbitacin I can suppress the growth of HCC cells, and the mechanism may be associated with its down-regulating the expressions of anti-apoptotic factors through STAT3 signaling pathway, and thereby inducing cell apoptosis.