The aim of the current study was to determine whether ventricular hypertrophy affects the delayed isoflurane preconditioning against myocardial ischemia-reperfusion (IR) injury. Transverse aortic constriction (TAC) was performed on male Sprague-Dawley rats to induce left ventricular (LV) hypertrophy, then sham-operated or hypertrophied rat hearts were subjected to isoflurane preconditioning (2.1% v/v, 1 h). 24 h after exposure, the hearts were isolated and perfused retrogradely by the Langendorff for 30 min (equilibration) followed by 40 min of ischemia and then 120 min of reperfusion. The hemodynamics, infarct size, apoptosis, nitric oxide synthase (NOS), cyclooxygenase-2 (COX-2), Cleaved Caspase-3 and production of NO were determined. We found that the hemodynamic parameters were all markedly improved during the reperfusion period and the myocardial infarct size and apoptosis was significantly reduced by delayed isoflurane preconditioning in sham-operated rats. However, such cardiac improvement induced by delayed isoflurane preconditioning was not observed in hypertrophied hearts. The expression of iNOS, COX-2 and NO was markedly enhanced, whereas Cleaved Caspase-3 activity was inhibited by delayed isoflurane preconditioning in sham-operated rats, a phenomenon was not found in TAC-control groups pretreated with isoflurane. Our results demonstrated that ventricular hypertrophy abrogated isoflurane-induced delayed cardioprotection by alteration of iNOS/COX-2 pathway.