Objective To explore whether microR-21 (miR-21) can alleviate doxorubicin (DOX)-induced cardiotoxicity and whether silent information regulator 1 (SIRT1) signaling pathway mediates the roles. Methods Neonatal rat cardiac myocytes were treated with DOX (1 μmol/L) to induce DOX myocardial toxicity model. The cardiomyocytes were randomized into 8 groups:control group, miR-21 group, miR-21 inhibitor group, DOX group, miR-21+DOX group, miR-21 inhibitor+DOX group, Sirtinol+miR-21+DOX group and Sirtinol+DOX group. The miR-21 mimics, miR-21 inhibitors and Sirtinol (SIRT1 inhibitor) were given at 24 h before DOX treatment. After treatment with DOX for 24 h, the cell viability, apoptosis rate, and the expression levels of apoptosis-related proteins and SIRT1 signaling pathway were detected. Results Compared with the control group, the cell viability, and the expression levels of Bcl-2 and SIRT1 were significantly decreased in the cardiomyocytes after treatment with DOX for 24 h, while the expression levels of Bax and cleaved Caspase-3, and apoptotic rate were significantly increased (P<0.05). Compared with the DOX group, miR-21 significantly increased cell viability and the expression levels of Bcl-2 and SIRT1, and significantly decreased the expression levels of Bax and cleaved Caspase-3 and apoptotic rate (P<0.05). Inhibiting SIRT1 signaling pathway could significantly weaken the protective effect of miR-21 on cardiomyocytes (P<0.05). Conclusion miR-21 can inhibit cardiomyocyte apoptosis, increase cell viability and alleviate DOX-induced cardiotoxicity by activating SIRT1 signaling pathway.