Activation of endothelial nitric oxide synthase (eNOS) in portal hypertensive (PHT) gastric mucosa leads to hyperdynamic circulation and increased susceptibility to injury. However, the signaling mechanisms for eNOS activation in PHT gastric mucosa and the role of TNF-α in this signaling remain unknown. In PHT gastric mucosa we studied (1) eNOS phosphorylation (at serine 1177) required for its activation; (2) association of the phosphatidylinositol 3-kinase (PI 3-kinase), and its downstream effector Akt, with eNOS; and, (3) whether TNF-α neutralization affects eNOS phosphorylation and PI 3-kinase–Akt activation. To determine human relevance, we used human microvascular endothelial cells to examine directly whether TNF-α stimulates eNOS phosphorylation via PI 3-kinase. PHT gastric mucosa has significantly increased (1) eNOS phosphorylation at serine 1177 by 90% (P < .01); (2) membrane translocation (P < .05) and phosphorylation (P < .05) of p85 (regulatory subunit of PI 3-kinase) by 61% and 85%, respectively; (3) phosphorylation (P < .01) and activity (P < .01) of Akt by 40% and 52%, respectively; and (4) binding of Akt to eNOS by as much as 410% (P < .001). Neutralizing anti–TNF-α antibody significantly reduced p85 phosphorylation, phosphorylation and activity of Akt, and eNOS phosphorylation in PHT gastric mucosa to normal levels. Furthermore, TNF-α stimulated eNOS phosphorylation in human microvascular endothelial cells. In conclusion, these findings show that in PHT gastric mucosa, TNF-α stimulates eNOS phosphorylation at serine 1177 (required for its activation) via the PI 3-kinase–Akt signal transduction pathway.