Abstract: Objective To test 32 traditional Chinese medicinal herbs with effects against osteosarcoma in vitro. Methods U2OS human osteosarcoma cell line was treated with the extracts of the Chinese herbs at various concentrations. The changes in cell proliferation in response to the treatment were examined by MTT assay, and the effects of these extracts against human osteosarcoma growth were compared. Morphological observation, flow cytometry and Annexin V were employed to detect the cell apoptosis after the treatments. Results According to the results of MTT assay, several of the 32 Chinese herbs, especially Venenum Bufonis and oxgall powder, were identified to produce growth inhibition against U2OS cells. Further study of the aqueous extracts of Venenum Bufonis and oxgall powder demonstrated their effects in inducing U2OS cell apoptosis, and Venenum Bufonis showed the strongest effect. In spite of the obvious growth inhibitory effect of oxgall powder, its extract induced cell apoptosis only at high concentrations. Conclusion Several of the traditional Chinese herbs, especially Venenum Bufonis and oxgall powder, may inhibit the growth of U2OS cell line, and the results of this study pave the way for further identification of the effective components in the herbs that inhibit osteosarcoma growth both in vivo and in vitro.