Pacing postconditioning (PPC) induces cardioprotection. The aim of this study was to determine the optimal pacing algorithm and possible influence of gender and diabetes on PPC. Unprotected regional ischemia for 30 min served as negative control and classical PPC (ten cycles of 30 s left ventricular pacing alternated with 30 s right atrial pacing) as positive control. Area at risk and infarct size were determined by blue dye and triphenyltetrazolium chloride staining. For achieving protection, the minimal number of PPC cycles was seven and the minimal duration of a PPC protocol was 200 s. The protective effect of PPC was comparable in male and female hearts, but no protection could be induced by PPC in diabetic hearts. PPC can provide myocardial protection when using at least seven cycles of ventricular pacing. PPC protection is independent of gender, but sensitive to experimental diabetes.