We have previously shown that insulin induces cardioprotection when administered at reperfusion after an ischemic insult. In the present study we have compared the possible role of Janus activating kinase 2 (JAK2)/ Signal transducer and activator of transcription 3 (STAT3) and glycogen syntase kinase 3 beta (GSK3B) in mediating cytoprotection under three different conditions: 1) During ischemic preconditioning (IPC), 2) when insulin was given as a PC-mimetic agent (InsPC) and 3) when SB (a GSK3B inhibitor) was given as a PC-mimetic agent (SBPC). Isolated perfused rat hearts were subjected to IPC (3x5min), InsPC (5 mU/ml; 3x5 min) or SBPC (3 ?M; SB administrated for 10 minutes prior to regional ischaemia) before taking them through 30 min of regional ischemia followed by 120 minutes of reperfusion ± AG (5 mM; STAT3 inhibitor). Risk zone (R) and infarct size (I) were determined with Evans blue and tetrazolium staining respectively. IPC, InsPC and SBPC treatment resulted in a significant reduction in infarct size as compared to controls (all p < 0.001). The protective effect of IPC and insulin was abolished by inhibition of the JAK/STAT signalling pathway, implying that cardioprotection offered by IPC and insulin is dependent on STAT3 activation. Inhibiting the JAK/STAT pathway did not abolish the cardioprotection afforded by SBPC, suggesting that STAT3 is upstream of GSK3B in the cardioprotective signalling pathway.