The clinical application of anthracyclines such as doxorubicin (DOX) is limited due to their cardiotoxicity. N6-methyladenosine (m 6 A) plays an essential role in numerous biological processes. However, the roles of m 6 A and m 6 A demethylase ALKBH5 in DOX-induced cardiotoxicity (DIC) remain unclear. In this research, DIC models were constructed using Alkbh5 -knockout (KO), Alkbh5 -knockin (KI), and Alkbh5 -myocardial-specific knockout (ALKBH5 flox/flox, αMyHC-Cre ) mice. Cardiac function and DOX-mediated signal transduction were investigated. As a result, both Alkbh5 whole-body KO and myocardial-specific KO mice had increased mortality, decreased cardiac function, and aggravated DIC injury with severe myocardial mitochondrial damage. Conversely, ALKBH5 overexpression alleviated DOX-mediated mitochondrial injury, increased survival, and improved myocardial function. Mechanistically, ALKBH5 regulated the expression of Rasal3 in an m 6 A-dependent manner through posttranscriptional mRNA regulation and reduced Rasal3 mRNA stability, thus activating RAS3, inhibiting apoptosis through the RAS/RAF/ERK signaling pathway, and alleviating DIC injury. These findings indicate the potential therapeutic effect of ALKBH5 on DIC., Competing Interests: Authors declare that they have no competing interests., (© 2023 The Authors.)