Activation and aggregation of platelets are key events in the pathophysiology of thrombotic diseases. There is increasing evidence that platelet-derived nitric oxide (NO) exerts important anti-platelet actions. Pyridoxine may have beneficial therapeutic effects in cardiovascular disease states, and has previously been shown to increase endothelial NO biosynthesis. The aims of the present study were firstly to determine in vitro whether pyridoxine can increase platelet NO synthesis, and secondly to investigate the mechanism by which it does this. Platelets isolated from blood taken from healthy subjects were treated with pyridoxine or vehicle. Platelet aggregation was measured by Born aggregometry. Intraplatelet cyclic guanosine-3',5'-monophosphate (cGMP, an index of bioactive NO) was measured by radioimmunoassay. Serine-1177-specific phosphorylation of NO synthase type 3 (NOS-3) and phosphorylation of protein kinase Akt were determined in platelets by Western blotting. Phosphatidylinositol 3-kinase (PI3K) activity in platelets was ascertained by homogeneous time-resolved fluorescence (HTRF) assay. Our results showed that pyridoxine largely inhibited the aggregation of platelets in response to adenosine diphosphate (ADP) or thrombin and increased bioactive NO. It also increased NOS-3 phosphorylation on serine-1177, and increased Akt serine phosphorylation. PI3K activity was augmented by pyridoxine, an effect inhibited by the specific PI3K antagonist LY294002. In conclusion, pyridoxine is effective in elevating platelet NO biosynthesis, through improving PI3K activity and hence downstream Akt phosphorylation, and in turn serine-1177 phosphorylation of NOS-3. These data reveal a novel mechanism by which NOS-3 activity can be regulated in platelets.