Sphingosine 1-phosphate (S1P) is released at sites of tissue injury and effects cellular responses through activation of G protein-coupled receptors. The role of S1P in regulating cardiomyocyte survival following in vivo myocardial ischemia-reperfusion (I/R) injury was examined by using mice in which specific S1P receptor subtypes were deleted. Mice lacking either S1P₂ or S1P₃ receptors and subjected to 1-h coronary occlusion followed by 2 h of reperfusion developed infarcts equivalent to those of wild-type (WT) mice. However, in S1P_2,3 receptor double-knockout mice, infarct size following I/R was increased by >50%. I/R leads to activation of ERK, JNK, and p38 MAP kinases; however, these responses were not diminished in S1P_2,3 receptor knockout compared with WT mice. In contrast, activation of Akt in response to I/R was markedly attenuated in S1P_2,3 receptor knockout mouse hearts. Neither S1P₂ nor S1P₃ receptor deletion alone impaired I/R-induced Akt activation, which suggests redundant signaling through these receptors and is consistent with the finding that deletion of either receptor alone did not increase I/R injury. The involvement of cardiomyocytes in S1P₂ and S1P₃ receptor mediated activation of Akt was tested by using cells from WT and S1P receptor knockout hearts. Akt was activated by S1P, and this was modestly diminished in cardiomyocytes from S1P₂ or S1P₃ receptor knockout mice and completely abolished in the S1P_2,3 receptor double-knockout myocytes. Our data demonstrate that activation of S1P₂ and S1P₃ receptors plays a significant role in protecting cardiomyocytes from I/R damage in vivo and implicate the release of S1P and receptor-mediated Akt activation in this process.