Background <p>Acute heart failure (AHF) is a life-threatening clinical syndrome due to impaired cardiac function. Ferroptosis has emerged as a contributor to cytotoxicity in cardiomyocytes. However, the functional interplay between USP8 and ferroptosis during AHF has not been investigated.</p> Methods <p>H9C2 rat cardiomyocytes were treated with doxorubicin (Dox) to establish an experimental model. Cell cytotoxicity was evaluated by measuring cell viability, LDH release, and cell death. Ferroptosis was assessed by analyzing Fe²⁺, lipid ROS, MDA, and GSH levels in treated cells. Immunoprecipitation (IP), Co-IP, and protein stabilization assays were performed to validate the USP8/murine double minute 4 (MDM4) interaction and the regulation of USP8 in MDM4. Expression of mRNA and protein was quantified by quantitative PCR and immunoblot analyses, respectively.</p> Results <p>USP8 and MDM4 were downregulated in Dox-exposed H9C2 cardiomyocytes. USP8 overexpression alleviated Dox-triggered cytotoxicity and cell death in H9C2 cardiomyocytes. Moreover, USP8 overexpression mitigated H9C2 cardiomyocyte ferroptosis induced by Dox. Mechanistically, USP8 stabilized MDM4 via deubiquitination. Inhibition of MDM4 counteracted the ability of USP8 overexpression to attenuate Dox-triggered cell death and ferroptosis in H9C2 cardiomyocytes.</p> Conclusion <p>Our findings indicate that USP8 overexpression protects H9C2 cardiomyocytes from Dox-induced ferroptosis by stabilizing MDM4 via deubiquitination.</p>