Abstract: CPUK02 (15-Oxosteviol benzyl ester) is a new semi-synthetic of stevioside. The anticancer activity was evaluated in several in vitro and in vivo models, and the mechanism of bioactivity studied. The anti-proliferation activity in vitro was tested by the MTT assay, which showed that CPUK02 strongly inhibited cell proliferation against a variety of human cancer cell lines. The human liver cancer cell line was significantly more sensitive than the normal liver cell line when CPUK02 was administered at low concentration. In several mouse bearing human cancer xenograft models, CPUK02 demonstrated comparable anti-tumor effects to fluorouracil (5-FU), but showed lower toxic effects. Annexin/PI double staining flow cytometry and DAPI staining experiments were used to observe the cellular morphological changes caused by CPUK02, which established that the compound induces cell apoptosis, and that the apoptotic effects appeared a dose dependent relationship. Western blot experiments were also carried out to explore the mechanism of this compound. It was found that this compound can activate the apoptosis signal cascade via the mitochondrial pathway in cells. These results indicate that CPUK02 may be a promising new compound with notable in vitro and in vivo anticancer activity. Further studies to investigate the effects on treating tumors are under way.