Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX-treated human OS cells.