Studies from our laboratory demonstrated the involvement of intrinsic apoptotic signaling in hyperpermeability following hemorrhagic shock (HS). Angiopoietin-1 (Ang-1), a potent inhibitor of hyperpermeability has recently been shown to inhibit apoptosis. The purpose of our study was to determine the effectiveness of Ang-1 in attenuating HS-induced hyperpermeability and its relationship to apoptotic signaling. Hemorrhagic shock was induced in rats by withdrawing blood to reduce the MAP to 40 mmHg for 1 hour followed by reperfusion. Mesenteric post-capillary venules were examined for changes in hyperpermeability utilizing intravital microscopy. Mitochondrial release of smac and cytochrome c were determined using western blot and ELISA respectively. Caspase-3 activity was determined by fluorometric assay. Parallel studies were performed in rat lung microvascular endothelial cell (RLMEC) monolayer utilizing HS serum and the pro-apoptotic BAK (BH3) peptide as inducers of hyperpermeability. In rats, Ang-1 (200 ng/ml) attenuated HS-induced hyperpermeability, versus HS group (p < 0.05). Ang-1 prevented HS-induced collapse of mitochondrial transmembrane potential (m), smac and cytochrome c release, and caspase-3 activity (p < 0.05). In RLMEC monolayer, HS serum and BAK (BH3) peptide both induced hyperpermeability that was inhibited by Ang-1 (p < 0.05). Angiopoeitin-1 attenuated HS and BAK (BH3) peptide induced-collapse of m, smac release, cytochrome c release, activation of caspase-3 and vascular hyperpermeability. In vivo BAK (BH3) induced vascular hyperpermeability that was attenuated by Ang-1 (p< 0.05). These findings suggest that the protective role of Ang-1 in microvascular endothelial barrier integrity involves the intrinsic apoptotic signaling cascade.