Objective: To assess whether myocardial fibrosis affects the protective efficiency of ischemic preconditioning (IPC) against myocardial ischemia/reperfusion injury (MIRI) in type 2 diabetic rats. Methods: Type 2 diabetic rat model was established. Fifty-four normal and 54 diabetic spragus-dawley (SD) rats were equally divided into 6 groups (n=18) using the random number table method: (1) Control group (C group); (2) Ischemia reperfusion injury (IRI) control group (IRI group); (3) IPC group; (4) Diabetic control group (DC group); (5) Diabetic IRI group (DIRI group); (6) Diabetic IPC group (DIPC group). After the reperfusion, blood samples were obtained for measuring serum concentrations of creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI) using enzyme-linked immunosorbent assay (ELISA). The myocardial infarction size (IS) was assessed by double staining method with Evan's blue and Triphenyl tetrazolium chloride (TTC), and the myocardial collagen volume fraction (CVF) and perivascular collagen area (PVCA) were assessed by Masson staining. Results: A stable and effective rat model with long-term diabetes was established in the current study. Compared with the normal rat groups, the CVF and PVCA significantly increased (all P<0.05) in the diabetic rat groups. The levels of CK-MB, cTnI and IS in the IPC group were (6.6±0.8) ng/ml, (0.5±0.1) ng/ml and (25.1±4.7) %, which showed significant decrease compared with (12.3±1.1) ng/ml, (1.2±0.3) ng/ml and (52.3±8.1) % in IRI group (all P<0.05). Among the diabetic rat groups, the CK-MB and cTnI levels in DIPC group were (11.5±0.9) and (1.1±0.1) ng/ml, apparently lower than the levels of (16.6±2.2) and (1.4±0.3) ng/ml in the DIRI group (both P<0.05). Compared with the IPC group, the IS, CK-MB and cTnI levels significantly increased in the DIPC group (all P<0.05). Conclusion: Myocardial fibrosis exists in rats with long-term type 2 diabetes, which weakens the protective effect of IPC on diabetes MIRI.