The Janus Activated Kinase (JAK) and Signal Transducers and Activators of Transcription (STAT) pathway is activated in the early and late phases of ischemic preconditioning (IPC) in normal myocardium. This pathway's role and the efficacy of IPC in hypertrophied hearts remain largely unknown. We hypothesized that activated STAT-3 (pSTAT-3) is necessary for effective IPC in pressure-overload hypertrophy. Male Sprague-Dawley rats eight weeks following thoracic aortic constriction (TAC) or sham operation underwent echocardiography and Langendorff perfusion. Randomized hearts were subjected to 30 minutes global ischemia, 120 minutes reperfusion with or without IPC in the presence or absence of the JAK-2 inhibitor, AG490. Functional recovery and STAT activation were assessed. TAC rats had a 31% increase in left ventricular mass (1347±58 mg v. 1028±43mg, TAC v. Sham, p<0.001), increased anterior and posterior wall thickness but no difference in ejection fraction compared to sham. In TAC, IPC improved end-reperfusion +dP/dt_max (4648±309 mmHg/s v. 2737±343 mmHg/s, IPC v. non-IPC, p<0.05) and -dP/dt_min (-2239±205 mmHg/s v. -1215±149 mmHg/s, IPC v. non-IPC, p<0.05). IPC increased nuclear pSTAT-1 and pSTAT-3 in sham but only pSTAT-3 in TAC. AG490 in TAC significantly attenuated +dP/dt_max (4648± 309 mmHg/s v. 3241±420 mmHg/s, IPC v. IPC+AG, p<0.05), -dP/dt_min(-2239±205 mmHg/s v. -1323±85 mmHg/s, IPC v. IPC+AG, p<0.05) and decreased only nuclear pSTAT-3. In myocardial hypertrophy, JAK-STAT signaling is important in ischemic preconditioning and exhibits a pattern of STAT activation distinct from non-hypertrophied myocardium. Limiting STAT-3 activation attenuates the efficacy of ischemic preconditioning in hypertrophy.