This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway.