Abstract: Background: Vascular homeostasis abnormalities may involve in doxorubicin induced cardiotoxicity. Methods: Enhanced cardiac miR‐320a expression, reduced cardiac microvessel density and impaired cardiac function were observed in mice treated by anthracycline doxorubicin. To further explore the role of miR‐320a in doxorubicin induced cardiotoxicity, microRNA mimics/inhibitor in vitro and rAAV administration in vivo were employed in mice. Results: Knockdown of miR‐320a not only resulted in enhanced proliferation and inhibited apoptosis in cultured endothelial cells, but also attenuated cardiac abnormalities induced by doxorubicin. On the contrary, overexpression of miR‐320a enhanced apoptosis in vitro, and aggravated vessel abnormalities in heart and subsequent cardiac dysfunction in mice. Furthermore, Western blot assays showed that VEGF‐A was a potential target of miR‐320a, which was verified by anti‐Ago2 co‐immunoprecipitation. Moreover, as same as miR‐320a, siRNA against VEGF‐A reinforced doxorubicin induced endothelial cells injury. Finally, the negative effects of miR‐320a on vascular homeostasis and cardiac function were alleviated by VEGF‐A re‐expression in doxorubicin treated mice. Conclusion: Our observations demonstrate that miR‐320a play important roles in doxorubicin induced cardiotoxicity via vessel homeostasis in heart and thus, inhibition of miR‐320a may be applied to the treatment of cardiac dysfunction induced by anthracycline.