Toona Sinensis leaf (TSL) aqueous extracts have been reported to have potent anti-proliferation effect on lung, melatonin, ovarian, oral, colon and liver cancer. Osteosarcoma is a malignant bone tumor that prevalents in adolescents. Although chemotherapy is effective in improvement of patients’ survival, the prognosis is not satisfiting. In this study, we investigated the effects of TS fractions on the proliferation of osteosarcoma cell lines (Saos-2, MG-63, and U2-OS), and the effects on normal human osteoblasts also compared. The results showed that treatment of fractions of TSL-1 to TSL-4 resulted in significant inhibition of cell proliferation than TSL-4P to TSL-7-treated groups. Furthermore, the anti-proliferation effect of TSL-2, TSL-3, and TSL-4 is similar to that of TSL-1. The results suggest that anticancer components exist in TSL-1 and it could be the most effective fraction inhibiting osteosarcoma proliferation. A treatment of osteosarcoma cells with TSL-1 also induced G2/M-phase arrest, and subsequently leading to cell death detected by Flow cytometry. TSL-1 mediated G2/M arrest was accompanied by the down-regulation of protein levels of cyclin B1 and cdc-2, and the up-regulation of p53, p21, and p-cdc25c. TSL-1-induced apoptosis was confirmed by cell morphology, Sub-G1 peak accumulation, cleavage of PARP, PI- Annexin-V double staining and TUNEL assay in MG-63. And the Western blot and RT-PCR results showed that TSL-1 treatment of MG-63 cells could induce apoptosis. Furthermore, our results also showed that the anti-proliferation mechanism might relate to cyclophilin A expression. In conclusion, our study indicates that TSL-1 inhibited cell proliferation of osteosarcoma, but not normal osteoblasts. We suggest that TSL-1 could be used as anticancer drug for alleviating osteosarcoma proliferation.