Sphingosine 1-phosphate (S1P) induces diverse biological responses in various tissues by activating specific G protein-coupled receptors (S1P₁–S1P₅ receptors). The biological signaling regulated by S1P₃ receptor has not been fully elucidated because of the lack of an S1P₃ receptor-specific antagonist or agonist. We developed a novel S1P₃ receptor antagonist, 1-(4-chlorophenylhydrazono)-1-(4-chlorophenylamino)-3,3-dimethyl- 2-butanone (TY-52156), and show here that the S1P-induced decrease in coronary flow (CF) is mediated by the S1P₃ receptor. In functional studies, TY-52156 showed submicromolar potency and a high degree of selectivity for S1P₃ receptor. TY-52156, but not an S1P₁ receptor antagonist [(R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester; VPC23019] or S1P₂ receptor antagonist [1-[1,3-dimethyl-4-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide; JTE013], inhibited the decrease in CF induced by S1P in isolated perfused rat hearts. We further investigated the effect of TY-52156 on both the S1P-induced increase in intracellular calcium ([Ca²⁺]_i) and Rho activation that are responsible for the contraction of human coronary artery smooth muscle cells. TY-52156 inhibited both the S1P-induced increase in [Ca²⁺]_i and Rho activation. In contrast, VPC23019 and JTE013 inhibited only the increase in [Ca²⁺]_i and Rho activation, respectively. We further confirmed that TY-52156 inhibited FTY-720-induced S1P₃ receptor-mediated bradycardia in vivo. These results clearly show that TY-52156 is both sensitive and useful as an S1P₃ receptor-specific antagonist and reveal that S1P induces vasoconstriction by directly activating S1P₃ receptor and through a subsequent increase in [Ca²⁺]_i and Rho activation in vascular smooth muscle cells.