Liver failure due to ischemia and reperfusion (IR) and subsequent acute kidney injury are significant clinical problems. We showed previously that liver IR selectively reduced plasma sphinganine-1-phosphate levels without affecting sphingosine-1-phosphate (S1P) levels. Furthermore, exogenous sphinganine-1-phosphate protected against both liver and kidney injury induced by liver IR. In this study, we elucidated the signaling mechanisms of sphinganine-1-phosphate-mediated renal and hepatic protection. A selective S1P(1) receptor antagonist blocked the hepatic and renal protective effects of sphinganine-1-phosphate, whereas a selective S1P(2) or S1P(3) receptor antagonist was without effect. Moreover, a selective S1P(1) receptor agonist, SEW-2871, provided similar degree of liver and kidney protection compared with sphinganine-1-phosphate. Furthermore, in vivo gene knockdown of S1P(1) receptors with small interfering RNA abolished the hepatic and renal protective effects of sphinganine-1-phosphate. In contrast to sphinganine-1-phosphate, S1P's hepatic protection was enhanced with an S1P(3) receptor antagonist. Inhibition of extracellular signal-regulated kinase, Akt or pertussis toxin-sensitive G-proteins blocked sphinganine-1-phosphate-mediated liver and kidney protection in vivo. Taken together, our results show that sphinganine-1-phosphate provided renal and hepatic protection after liver IR injury in mice through selective activation of S1P(1) receptors and pertussis toxin-sensitive G-proteins with subsequent activation of ERK and Akt.