Biochanin A has been reported to be associated to tumour cell proliferation, apoptosis and drug resistance in hepatocellular carcinoma, prostate cancer, and colorectal cancer, etc, while the relation of biochanin A on osteosarcoma is not clear. Hence, in this study, we examined the effects of biochanin A treatment on osteosarcoma cell lines MG63 and U2OS on proliferation, apoptosis, invasion and migration. We then investigated the involved molecular mechanism and found a time- and dose-dependent inhibition of cell viability in MG63 and U2OS cells with biochanin A treatment. Under the same circumstances, an increased ratio of cells in G0/G1 phase but a decreased ratio of cells in G2/M phase was observed. In addition, after biochanin A treatment, apoptotic rates clearly increased and decreased migration and invasion ability were observed in MG63 and O2OS cells. Meanwhile, relevant genes involved in cell proliferation, apoptosis, invation and migration demonstrated altered expressions in MG63 and U2OS cells. The present study supports the assumption that biochanin A has suppressive effects on osteosarcoma through regulating cell proliferation, apoptosis, invasion, and migration.