Phosphoinositide 3-kinase (PI3-K) and Akt play important roles in platelet activation. However, the downstream mechanisms for their roles are unclear. We have recently shown that nitric oxide (NO) synthase 3 (NOS3) and cGMP-dependent protein kinase stimulate platelet secretion and aggregation. Here we show that PI3-K-mediated Akt activation plays a critical role in agonist-stimulated platelet NO synthesis and cGMP elevation. Agonist-induced elevation of NO and cGMP was inhibited by Akt inhibitors and reduced in Akt-1 knockout platelets. Akt-1 knockout or Akt inhibitor-treated platelets showed reduced platelet secretion and aggregation in response to low concentrations of agonists, which can be reversed by low concentrations of sodium nitroprusside (SNP) or cGMP analogs. Similarly, PI3-K inhibitors diminished elevation of cGMP and also inhibited platelet secretion and the second-wave platelet aggregation, which was also partially reversed by cGMP analogs and by SNP. These results indicate that the NO-cGMP pathway is an important downstream mechanism mediating PI3-K and Akt signals leading to platelet secretion and aggregation. Conversely, the PI3-K-Akt pathway is a major upstream mechanism responsible for activating the NO-cGMP pathway. Thus, this study delineates a novel platelet activation pathway involving sequential activation of PI3-K, Akt, NOS3, sGC, and cGMP-dependent protein kinase.