Objective To explore the potential mechanism of ferroptosis regulated by VDAC3 in sepsis-induced myocardial injury. Methods A total of 20 male C57BL/6J mice (7~8 weeks old) were randomly divided into 2 groups, respectively named Sham group and CLP group, with 10 mice in each group. Sepsis was induced by the cecal ligation and puncture (CLP) . The levels of IL-6, TNF-α, CK-MB, and cTnT in the serum were detected by enzyme-linked immunosorbent assay (ELISA); the pathological changes of the heart tissue were observed by hematoxylin and eosin (H&E) staining; the structural and functional changes of the heart were evaluated by echocardiography; the changes of total glutathione , reduced glutathione (GSH), oxidized glutathione (GSSG), and malondialdehyde (MDA) in the heart tissue were detected by spectrophotometry; the morphological structure of mitochondria in mouse cardiomyocytes was observed by transmission electron microscope; the expression levels of IL-6, IL-1β, VDAC3, GPX4, SLC7A11, LCN2 and PTGS2 mRNA were detected by real-time quantitative polymerase chain reaction (qRT-PCR); and the expression levels of VDAC3, GPX4 and SLC7A11 proteins were detected by Western blot. Results The levels of IL-6, TNF-α, CK-MB and cTnT in the CLP group mice were significantly higher than that in the Sham group (P<0.05). After 24 h of CLP, it was observed that myocardial fibers were torn, the ventricular wall was thickened and edematous, and mitochondrial membrane was ruptured, mitochondrial cristae were broken or even disappeared. Moreover, the GSH levels were significantly reduced in the CLP group compared with the Sham group (P<0.05) ; the lipid peroxide MDA were increased in the CLP group(P<0.05). Furthermore, compared with the Sham group, the levels of VDAC3, GPX4 and SLC7A11 mRNA and protein were all lowered (P<0.05) in the CLP group, and the expression levels of IL-6, IL-1β, LCN2 and PTGS2 mRNA were increased (P<0.05). Conclusions VDAC3 expression decreases in myocardial injury, and it may participate in the occurrence of sepsis-induced myocardial injury by regulating ferroptosis.