The purpose of this study was to observe the functions of preconditioning with endoplasmic reticulum stress (ERS) whether alleviated heart ischemia/reperfusion injury (HI/RI) via modulating IRE1/ATF6/RACK1/PERK and PGC-1α expressions in diabetes mellitus (DM) or not. Diabetic rats were pretreated with 0.6 mg/kg tunicamycin (TM, 0.6 mg/kg tunicamycin was administered via intraperitoneal injection 30 minutes prior to the I/R procedures), and then subjected to 45 minutes of ischemia and 3 hours of reperfusion. Blood and myocardial tissues were collected, myocardial pathological injuries were investigated, serum creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT) levels were measured, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximum rate of left ventricular pressure rise (+dp/dt_max) and maximum rate of left ventricular pressure drop (-dp/dt_max) were evaluated, reactive oxygen species (ROS) and caspase-3 levels were observed, ΔΨm level and ROS expression were measured, and activated transcript factor 6 (ATF6), receptor for activated C kinase 1 (RACK1), PRK-like ER kinase (PERK), glucose regulated protein 78 (GRP78) and peroxisome proliferator-activated receptor γ co-activator 1-α (PGC-1α) expressions were assessed. The TM ameliorated the pathological damages, reduced myocardial oxidative stress damages, restrained apoptosis, and upregulated the expressions of ATF6, RACK1, PERK, GRP78 and PGC-1α compared with those of the ischemia/reperfusion (I/R) group in DM. This study suggested the preconditioning with endoplasmic reticulum stress (TM) strategy that could enhance protection against HI/RI in DM in clinical myocardial diseases.