Recent studies demonstrated a role of Sphingosine-1-phosphate (S1P) in protection against the stress of ischemia/reperfusion injury (I/RI). In experiments reported here, we have investigated the signaling through the S1P cascade by FTY720, a sphingolipid drug candidate displaying structural similarity to S1P, underlying the S1P cardio-protective effect. In ex vivo rat heart and isolated sino-atrial node models, FTY720 significantly prevented I/RI induced arrhythmic events including premature ventricular beats, VT and sinus bradycardia as well as A-V conduction block. Real-time PCR and Western blot analysis demonstrated the expression of the S1P receptor transcript pools and corresponding proteins including S1P1, S1P2 and S1P3 in tissues dissected from sino-atrial node, atrium and ventricle. FTY 720 (25 nM) significantly blunted the depression of the levels of phospho-Pak1 and phospho-Akt with ischemia and with reperfusion. There was a significant increase in phospho-Pak1 levels by 35%, 199%, 205% after 5, 10 and 15 mins of treatment with 25 nM FTY720 compared with control non-treated myocytes. However, there was no significant difference in the levels non-phospho-Pak1 expression between non-treated and FTY720 treated. Phospho-Akt levels were increased by 44%, 63%, and 61% after 5, 10 and 15 min of treatment with 25 nM FTY720 respectively. Our data provide the first evidence that FTY720 prevents the arrhythmias induced by I/RI, and indicate its potential significance as an important and new agent protecting against ischemia/reperfusion induced arrhythmias. The cardio-protective effect of FTY720 is likely to involve activation of signalling through the Pak1 and Akt cascade.