Objective To study the mechanism underlying cucurbitacin I (JSI-124) inducing cell apoptosis in human hepatoma HepG2 cells. Methods HepG2 cells were exposed to 0.01, 0.10, 1.00 and 10.00 μmol/L JSI-124 for 24, 48 and 72 hours. Cell proliferation was detected by CCK-8 assay; the nuclear morphological changes were observed by Hoechst 33258 staining; the formation of tumor cell colonies was visualized by violet staining; and cell apoptosis was detected by annexin V-FITC/PI double staining combined with flow cytometry. Furthermore, the mRNA expression levels of p53 and its downstream Bax, Fas and MDM2 genes were measured by quantitative real-time PCR, and the protein levels of P53 and activated caspase-3 were evaluated by Western blotting. Results JSI-124 inhibited the proliferation and induced Hoechst 33258-stained chromatin condensation in HepG2 cells in a concentration- and time-dependent manner. Flow cytometry revealed that 1.00 μmol/L JSI-124 treatment increased the apoptotic rate significantly in HepG2 cells compared with the control cells. Furthermore, JSI-124 significantly enhanced the mRNA expressions of p53 and its downstream apoptotic factors, including Bax and Fas, but did not change the gene expression of the p53 tumor suppressor, MDM2. The 48-hour treatment of JSI-124 in HepG2 cells significantly increased the levels of p53 and cleaved caspase-3 proteins. Conclusion JSI-124 induces the apoptosis of HepG2 cells through the activation of p53 and its downstream pro-apoptotic factors.