Abstract The study was designed to investigate the effect of progesterone and its gender based variation on myocardial ischemia/reperfusion (I/R) injury in rats. Adult Sprague Dawley rats were divided into vehicle treated reperfusion injury group male (I/R-M), female (I/R-F), ovariectomised (I/R-OVR) and progesterone treatment (I/R-M + PG, I/R-F + PG, I/R-OVR + PG) groups, respectively. I/R injury was produced by occluding the left descending coronary artery (LCA) for 1 h and followed by re-opening for 1 h. Progesterone (2 mg kg − 1 i.p.) was administered 30 min after induction of ischemia. Hemodynamic parameters (± d p/d t, MAP), heart rate, ST-segment elevation and occurrence of ventricular tachycardia (VT) were measured during the I/R period. The myocardial infarct area, oxidative stress markers, activities of myeloperoxidase (MPO) and creatine kinase (CK) were determined after the experiment along with the assessment of the effect on apoptotic activity by using DNA fragmentation analysis. Histological observations were carried out on heart tissue. Treatment with progesterone significantly ( P < 0.05) reduced infarct area, lipid peroxidation (LPO) level and activity of MPO in females (I/R-F + PG) as compared to ischemic females (I/R-F). Progesterone significantly ( P < 0.001, P < 0.05) inhibited serum CK activity and incidences of VT in female rats. Superoxide dismutase (SOD) activity, reduced glutathione (GSH) levels were significantly elevated ( P < 0.05) in I/R-F + PG group. Internucleosomal DNA fragmentation was less in I/R-F + PG group when compared to I/R-F group. The ischemic male and ovariectomised (I/R-M and I/R-OVR) counterparts did not show any significant change after progesterone treatment. In conclusion, the cardioprotective effect of progesterone on myocardial I/R injury induced damage is based on gender of the animal. The protective effect could be mediated by attenuation of inflammation and its possible interaction with endogenous estrogen.