Abstract: To study the synergistic anti-tumor effects of doxorubicin and axitinib in combination, and investigate the underlying mechanism, we performed the MTT cytotoxic assay and tumor spheroids inhibition to investigate in vitro using A549 cells. The cell internalization, cell cycle distribution and DNA ladder experiments were performed to study the synergistic mechanisms. A549 xenograft was established in nude mice and adopted to study the in vivo anti-tumor effect of doxorubicin and axitinib. Results showed that combination of doxorubicin and axitinib exerted significantly higher cytotoxicity than each single drug, and induced a synergistic effect on tumor spheroids growth inhibition. The combination achieved the highest tumor growth suppression in vivo in the A549 xenograft. The combination of axitinib and doxorubicin exhibited the best anti-tumor effects both in vitro and in vivo than each single drug.