Phospholipase D (PLD), phosphatidylinositol 3-kinase (PI 3-kinase) and Akt are known to be involved in cellular signaling related to proliferation and cell survival. In this report, we provide evidence that PLD links sphingosine 1-phosphate (S1P)- induced activation of the G protein-coupled EDG3 receptor to stimulation of PI 3-kinase and its downstream effector Akt in Chinese hamster ovary (CHO) cells. S1P stimulation of EDG3-overexpressing CHO cells, but not vector-transfected cells, induced activation of PLD, PI 3-kinase, and Akt in a time- and dose-dependent manner. The Akt phosphorylation was prevented by the PI 3-kinase inhibitors wortmannin and LY294002, indicating that the Akt activation was dependent on PI 3-kinase. S1P-induced activation of PI 3-kinase and Akt was abrogated by 1-butanol, which inhibited S1P-induced accumulation of phosphatidic acid (PA) by serving as a phosphatidyl group acceptor in the transphosphatidylation reaction catalyzed by PLD, whereas it was not inhibited by 2-butanol without such an action. Coexpression of the wild-type PLD2 with myc-Akt resulted in increased Akt activation in response to S1P. In contrast, co-expression of a catalytically inactive mutant of PLD2 eliminated the S1P-induced Akt activation. The treatment of EDG3-expressing CHO cells with exogenous Streptomyces chromofuscus PLD, which caused an accumulation of PA, resulted in increases in PI 3-kinase activity and the phosphorylation of Akt, the latter of which was completely abolished by LY294002. Furthermore, S1P-induced membrane ruffling, which was dependent on PI 3-kinase and Rac, was inhibited by 1-butanol, but not 2-butanol. These results demonstrate that PLD participates in the activation of PI 3-kinase and Akt in stimulation of EDG3 receptor.