Macrophage recruitment to sites of inflammation is an essential step in host defense. However, the mechanisms preventing excessive accumulation of macrophages remain relatively unknown. The lysophospholipid sphingosine 1-phosphate (S1P) promotes T and B cell egress from lymphoid organs by acting on S1P receptor 1 (S1P 1 R). More recently, S1P 5 R was shown to regulate NK cell mobilization during inflammation, raising the possibility that S1P regulates the trafficking of other leukocyte lineages. In this study, we show that S1P 2 R inhibits macrophage migration in vitro and that S1P 2 R-deficient mice have enhanced macrophage recruitment during thioglycollate peritonitis. We identify the signaling mechanisms used by S1P 2 R in macrophages, involving the second messenger cAMP and inhibition of Akt phosphorylation. In addition, we show that the phosphoinositide phosphatase and tensin homolog deleted on chromosome 10, which has been suggested to mediate S1P 2 R effects in other cell types, does not mediate S1P 2 R inhibition in macrophages. Our results suggest that S1P serves as a negative regulator of macrophage recruitment by inhibiting migration in these cells and identify an additional facet to the regulation of leukocyte trafficking by S1P.