Imatinib mesylate (IMA) belonging to the selective tyrosine kinase inhibitor family has been proven to induce cardiotoxic effects along with therapeutic strategies. Nrf2-dependent ferroptosis has been implicated in the cardiotoxicity induced by IMA. The present study was designed to investigate the protective effects of berberine hydrochloride (Ber) on cardiac injuries induced by IMA and to explore its potential mechanisms. In H9c2 cells, cell viability, the generation of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and labile iron pool (LIP) levels were measured. In a mouse model of IMA-induced cardiomyopathy, serum biomarkers and cardiac tissues were examined. A western blot assay was performed to evaluate the expression of ferroptosis-related proteins in vitro and in vivo. Our results indicated that Ber increased cell viability and MMP and decreased cellular ROS and iron levels in comparison to the IMA group of H9c2 cells. In mice, Ber significantly improved cardiac status and attenuated the level of ferroptosis biomarkers including malonaldehyde (MDA) and iron content. Additionally, Ber downregulated the expression of transferrin receptor (TfR) and P53 and upregulated the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase-1 (NQO1), ferritin heavy chain-1 (FTH1), and glutathione peroxidase 4 (GPX4) in H9c2 cells and mice. The present data indicated that Ber has the potential to protect against IMA-induced cardiotoxicity, partly via inhibiting Nrf2-dependent ferroptosis.