Akt plays a pivotal role in cell survival and tumorigenesis. We investigated the potential interaction between sphingosine-1-phosphate (S1P) and platelet-derived growth factor (PDGF) in the Akt signaling pathway. Using mouse embryonic fibroblasts (MEFs) from S1P receptor knockout mice, we show here that S1P3 was required for S473 phosphorylation of Akt by S1P. In addition, S1P-stimulated activation of Akt, but not ERK, was blocked by a PDGF receptor (PDGFR)-specific inhibitor, AG1296, suggesting a S1P3-mediated specific crosstalk between the Akt signaling pathways of S1P and PDGFR in MEFs. We investigated this crosstalk under different conditions and found that both Akt and ERK activation induced by S1P, but not lysophosphatidic acid (LPA), in HEY ovarian cancer cells required PDGFR but not epidermal growth factor receptor (EGFR) or insulin-like growth factor-I receptor (IGFR). Importantly, S1P induced a Gi-dependent tyrosine phosphorylation of PDGFR in HEY cells. This dependence on PDGFR in S1P-induced Akt activation was also observed in A2780, T47D, and HMEC-1 cells (which express S1P3), but not in PC-3 or GI-101A cells (which do not express S1P3), further supporting that S1P3 mediates the crosstalk between S1P and PDGFR. This is the first report demonstrating a unique interaction between S1P3 and PDGFR, in addition to demonstrating a specific role for S1P3 in S1P-induced Akt activation.