Objective This study aimed to investigate the ameliorative effect and potential mechanism of intermittent fasting ( IF) on myocardial injury in diabetic mice. Methods Eight-week-old male db / db mice were used to establish a diabetic myocardial injury model and randomly divided into db / db group and IF group, with db / m mice as the db / m group. The IF group underwent 24-hour fasting / 24-hour feeding alternation for 8 weeks. For in vitro experiments, H9c2 cell injury was induced by high glucose, and cells were randomly divided into control group, high glucose (HG) group, HG+IF group, and HG+IF+PI3K inhibitor (LY294002) group. Cardiac function was assessed by echocardiography. Serum levels of lactate dehydrogenase ( LDH) and creatine kinase-MB ( CK-MB) were measured by enzyme-linked immunosorbent assay. Myocardial histopathology was evaluated using hematoxylin-eosin (HE) and Masson’s trichrome staining. Protein expression of key signaling molecules was analyzed by Western blot. Results Compared with db / db group, IF significantly reduced random blood glucose and body weight (P<0. 05,P<0. 01, P<0. 001), increased left ventricular fractional shortening ( LVFS), ejection fraction ( LVEF) and E/ A ratio (P<0. 001), and decreased end-diastolic / end-systolic ventricular wall thickness, ventricular volume and heartto-body weight ratio (HW/ BW, P<0. 05, P<0. 001). IF also decreased serum lactate dehydrogenase (LDH) activity and creatine kinase-MB (CK-MB) level (P<0. 001), alleviated cardiomyocyte hypertrophy and interstitial collagen deposition, and lowered myocardial collagen volume fraction (P<0. 001). The protein levels of p-PI3K and p-AKT in myocardial tissue were significantly upregulated by IF (P<0. 01, P<0. 001). In vitro, IF markedly improved the survival rate and viability of HG-injured cardiomyocytes (P<0. 01, P<0. 001), and reduced LDH activity and CKMB level in cell supernatant (P<0. 01, P<0. 001). After inhibitor intervention, the above protective effects of IF were reversed, with decreased cell survival and viability and restored levels of myocardial injury markers (P<0. 01,P<0. 001). Conclusions IF ameliorates diabetic myocardial injury by activating the PI3K/ AKT signaling pathway.