Background: Previous studies have demonstrated that G-protein-coupled receptor kinase interacting-1 protein (GIT1) associates with eNOS to regulate nitric oxide production in sinusoidal endothelial cells (SECs). Here, we hypothesized that GIT1's tightly-associated binding partner, b-PIX (p21-activated kinase-interacting exchange factor beta, ARHGEF7) is specifically important in regulation of eNOS activity. Methods: We examined b-PIX expression in normal rat liver by immunohistochemistry and explored b-PIX protein-protein interactions using immunoprecipitation and immunoblotting. The role of b-PIX in regulating eNOS enzymatic activity was studied in GIT1-deficient SECs. Finally, structural analysis of interaction sites in GIT1 and β-PIX required to regulate eNOS activity were mapped. Results: b-PIX was expressed primarily in SECs in normal liver, and was either absent or expressed at extremely low levels in other liver cells (stellate cells, Kupffer cells, hepatocytes). β-PIX interacted with GIT1 and eNOS to form a trimolecular signaling module in normal SECs, and was important in stimulating eNOS activity. Of note, GIT1-β-PIX interaction led to synergistic enhancement of eNOS activity, and β-PIX-driven increase in eNOS activity was GIT1 dependent. Disruption of β-PIX or GIT1 in normal SECs using β-PIX siRNA or GIT1-deficient SECs led to reduced eNOS activity. Finally, specific GIT1 domains (SHD and SLD, aa 331 to 596) and the β-PIX C terminal (aa 496 to 555) appeared to be critical in the regulation eNOS activity. Conclusions: The data indicate that b-PIX regulates eNOS phosphorylation and function in normal SECs, and highlight the importance of the GIT1/b-PIX/eNOS trimolecular complex in normal liver SEC function.