--- - |2 Nucleolin is a multifunctional protein and participates in many important biological processes. Our previous study found that nucleolin protects the heart against myocardial ischemia–reperfusion injury. In this study, we aimed to investigate the role of nucleolin in doxorubicin (DOX)‐induced cardiotoxicity. The expression pattern of nucleolin in hearts subjected to DOX injury was investigated, and we found that administration of DOX induced nucleolin expression significantly in vivo and in vitro. Gene transfection and RNA interference approaches were used in cardiomyocytes to investigate the function of nucleolin. Nucleolin overexpression protects cardiomyocytes against DOX‐induced injury. Nucleolin‐ablated cardiomyocytes become susceptible to the injury induced by DOX. The hearts of cardiac‐myocyte‐specific nucleolin transgenic mice are more resistant to DOX injury. Furthermore, nucleolin upregulates microRNA(miRNA)‐21 expression in vivo and in vitro, and the miRNA‐21 inhibitor negates the protective effect of nucleolin against injury induced by DOX. These results have demonstrated that nucleolin is involved in the regulation of DOX‐induced cardiac injury and dysfunction via the regulation of miRNA‐21 expression, and may be a novel therapeutic target for DOX‐induced cardiotoxicity. - Journal of Cellular Physiology, Volume 233, Issue 12, Page 9516-9525, December 2018.