Phytochemicals used in cancer therapy and prevention are an important source. Betulinic acid (BetA), a lupine-type pentacyclic triterpenoid saponin from plants, has shown anti-tumor activity in some cell lines in previous studies. In this paper, its anti-tumor effect and the possible mechanisms were investigated in U14 tumor-bearing mice. The results showed that BetA (100 mg/kg and 200 mg/kg) effectively suppressed tumor growth in vivo. Compared with the control group, BetA significantly improved the levels of IL-2 and TNF-alpha in tumor-bearing mice and increased the number of CD4+ lymphocytes subsets, as well as the ratio of CD4+/CD8+ at a dose of 200 mg/kg. Furthermore, treatment with BetA induced cells apoptosis in dose-dependent manner in tumor bearing mice, and inhibited the expression of Bcl-2 and Ki-67 protein while upregulated the expression of caspase-8 protein. The mechanisms by which BetA exerted anti-tumor effects might involve the induction of tumor cell apoptosis. This process is also related to improvement of body's immune response.