Clinical application of doxorubicin (DOX), an anthracycline antibiotic with potent anti- tumor effects, is limited because of its cardiotoxicity. However, its pathogenesis is still not entirely understood. The aim of this paper was to explore the mechanisms and new drug targets to treat DOX-induced cardiotoxicity. The in vitro model on H9C2 cells and the in vivo models on rats and mice were developed. The results showed that DOX markedly decreased H9C2 cell viability, increased the levels of CK, LDH, caused histopathological and ECG changes in rats and mice, and triggered myocardial oxidative damage via adjusting the levels of intracellular ROS, MDA, SOD, GSH and GSH-Px. Total of 18 differentially expressed microRNAs in rat heart tissue caused by DOX were screened out using microRNA microarray assay, especially showing that miR-140-5p was significantly increased by DOX which was selected as the target miRNA. Double-luciferase reporter assay showed that miR-140-5p directly targeted Nrf2 and Sirt2, as a result of affecting the expression levels of HO-1, NQO1, Gst, GCLM, Keap1 and FOXO3a, and thereby increasing DOX-caused myocardial oxidative damage. In addition, the levels of intracellular ROS were significantly increased or decreased in H9C2 cells treated with DOX after miR-140-5p mimic or miR-140-5p inhibitor transfection, respectively, as well as the changed expression levels of Nrf2 and Sirt2. Furthermore, DOX- induced myocardial oxidative damage was worsened in mice treated with miR-140-5p agomir, and however the injury was alleviated in the mice administrated with miR-140-5p antagomir. Therefore, miR-140-5p plays an important role in DOX-induced cardiotoxicity by promoting myocardial oxidative stress via targeting Nrf2 and Sirt2. Our data provide novel insights for investigating DOX-induced heart injury. In addition, miR-140-5p/ Nrf2 and miR-140-5p/Sirt2 may be the new targets to treat DOX-induced cardiotoxicity.