Sphingosine-1-phosphate (S1P) participates in inflammation; however, its role in leukocyte rolling is still unclear. Here we use intravital microscopy in inflamed mouse cremaster muscle venules and human endothelial cells to show that S1P contributes to P-selectin-dependent leukocyte rolling through endothelial S1P receptor 3 (S1P₃) and Gα_q, PLCβ and Ca²⁺. Intra-arterial S1P administration increases leukocyte rolling, while S1P₃ deficiency or inhibition dramatically reduces it. Mast cells involved in triggering rolling also release S1P that mobilizes P-selectin through S1P₃. Histamine and epinephrine require S1P₃ for full-scale effect accomplishing it by stimulating sphingosine kinase 1 (Sphk1). In a counter-regulatory manner, S1P₁ inhibits cAMP-stimulated Sphk1 and blocks rolling as observed in endothelial-specific S1P₁^−/− mice. In agreement with a dominant pro-rolling effect of S1P₃, FTY720 inhibits rolling in control and S1P₁^−/− but not in S1P₃^−/− mice. Our findings identify S1P as a direct and indirect contributor to leukocyte rolling and characterize the receptors mediating its action.