Sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, is generated and released at sites of tissue injury in the heart and can act on S1P₁, S1P₂, and S1P₃ receptor subtypes to affect cardiovascular responses. We established that S1P causes little phosphoinositide hydrolysis and does not induce hypertrophy indicating that it does not cause receptor coupling to G_q. We previously demonstrated that S1P confers cardioprotection against ischemia/reperfusion by activating RhoA and its downstream effector PKD. The S1P receptor subtypes and G proteins that regulate RhoA activation and downstream responses in the heart have not been determined. Using siRNA or pertussis toxin to inhibit different G proteins in NRVMs we established that S1P regulates RhoA activation through Gα₁₃ but not Gα₁₂, Gα_q, or Gα_i. Knockdown of the three major S1P receptors using siRNA demonstrated a requirement for S1P₃ in RhoA activation and subsequent phosphorylation of PKD, and this was confirmed in studies using isolated hearts from S1P₃ knockout (KO) mice. S1P treatment reduced infarct size induced by ischemia/reperfusion in Langendorff perfused wild-type (WT) hearts and this protection was abolished in the S1P₃ KO mouse heart. CYM-51736, an S1P₃-specific agonist, also decreased infarct size after ischemia/reperfusion to a degree similar to that achieved by S1P. The finding that S1P₃ receptor- and Gα₁₃-mediated RhoA activation is responsible for protection against ischemia/reperfusion suggests that selective targeting of S1P₃ receptors could provide therapeutic benefits in ischemic heart disease.