Macrophages, the most plastic cells in the haematopoietic system, are found in all tissues and show great functional heterogeneity. Sphingosine 1-phosphate (S1P)/S1P receptors (S1PRs) system is widely involved in the process of inflammatory disease, whereas little evidence concerning its role in functional macrophage polarization is available. Thus, the present study was designed to evaluate the effects of S1P/S1PRs on functional polarization of macrophage in mouse bone marrow (BM)-derived monocyte/macrophages (BMMs). For the detection of M1 macrophage markers, such as CD86, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1/chemokine (CC motif) ligand (CCL) 2, nitric oxide synthase (NOS) 2, and macrophage inflammatory protein (MIP)-1β, RT-qPCR and cytometric bead array (CBA) were performed in cultured primary BMMs after the treatment with selective S1PR 2/3 antagonists or specific S1PRs siRNA. Western blotting and immunofluorescence were used for the detection of phosphorylation of JNK1/2. BMMs expressed S1PR 1-3 and interestingly, S1PR 2/3, but not S1PR 1, mediates S1P-induced M1 macrophage polarization of BMMs as their siRNA or antagonists reduced M1 genes’ expression. We found that PTX (inhibitor of G (α) i/o), LY294002 (inhibitor of PI3K) or SP600125 (inhibitor of JNK1/2) prevented up-regulation of M1 genes expression mediated by S1P/S1PR 2/3 signal, and S1P-induced JNK phosphorylation was inhibited by antagonists of S1PR 2/3, PTX or LY294002. Collectively, our results demonstrate that S1P/S1PR 2/3 plays a key role in regulating M1 type polarization of BMMs and acts by activating G (α) i/o/PI3K/JNK signaling pathway, with potential implications for new approaches to inflammatory liver disease therapy.