Effects of emodin (EMD) treatment on mitochondrial ATP generation capacity and antioxidant components as well as susceptibility to ischemia-reperfusion (I-R) injury were examined in male and female rat hearts. Isolated-perfused hearts prepared from female rats were less susceptible to I-R injury than those of male rats. I-R caused significant decreases in ATP generation capacity and reduced glutathione (GSH) and alpha-tocopherol (alpha-TOC) levels as well as glutathione reductase, Se-glutathione peroxidase and Mn-superoxide dismutase (SOD) activities. The lower susceptibility of female hearts to myocardial I-R injury was associated with higher levels of GSH and alpha-TOC as well as activity of SOD than those of male hearts. EMD treatment at 3 daily doses (0.6 or 1.2 mmol/kg) could enhance myocardial mitochondrial ATP generation capacity and antioxidant components in both male and female rat hearts, but it only significantly protected against I-R injury in female hearts. Treatment with a single dose of EMD invariably enhanced mitochondrial antioxidant components and protected against I-R injury in both male and female hearts. The gender-dependent effect of EMD treatment at multiple doses may be related to the differential antioxidant response in the myocardium and/or induction of drug metabolizing enzymes in the liver.