【Abstract】 Objective This study aimed to investigate the ameliorative effect and potential mechanism of Intermittent Fasting (IF) on myocardial injury in diabetic mice. Methods Herein, 8-week-old male db/db mice were used as the the in vivo?diabetic myocardial injury model , which were randomly assigned to the model group and the intermittent fasting (IF)-treated group. Age-matched db/m mice served as the non-diabetic control group (Con). The IF group underwent an alternating 24-hour fast/24-hour feed cycle for 8 weeks, while the Con and model groups had?ad libitum?access to food. Cardiac function was assessed by echocardiography assay. Serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were measured by ELISA. Myocardial histopathology was evaluated using Hematoxylin & Eosin (H&E) and Masson's trichrome staining. Protein expression of key signaling molecules was analyzed by Western blot. An?in vitro?model was generated by exposing H9c2 cardiomyoblasts to high glucose (HG). Results Compared with the control group, the model group exhibited significantly increased random blood glucose (RBG) and body weight (BW) (P?< 0.05), which were both significantly reduced by intermittent fasting (IF) intervention (P?< 0.05). Cardiac function assessment revealed that the model group had significantly decreased left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) (P?< 0.001), along with reduced left ventricular posterior wall thickness at end-diastole (LVPWd) and end-systole (LVPWs) (P?< 0.05) ，left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were significantly increased (P < 0.01，P < 0.001), while the E/A ratio was significantly decreased (P < 0.01) and a lower heart weight/body weight ratio (HW/BW) (P?< 0.001). IF treatment significantly attenuated myocardial injury, as evidenced by reduced serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) activity (P?< 0.0001), and inhibited cardiac collagen deposition (P?< 0.0001). Mechanistically, IF significantly upregulated the activation of the PI3K/AKT signaling pathway?in vivo, indicated by increased ratios of p-PI3K (P?< 0.001) and p-AKT (P?< 0.01) in myocardial tissue, and similarly elevated these ratios in high glucose-treated cardiomyocytes?in vitro?(P?< 0.01). Conclusion IF improved diabetic myocardial function and structure, and alleviated myocardial injury by activating the PI3K/AKT signaling pathway, providing the new experimental evidence and potential strategies for the prevention and treatment of diabetic cardiac complications.