Excessive microvascular permeability is a serious complication following hemorrhagic shock and resuscitation (HSR). S1P has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and HSR. In the current study, we tested the hypothesis that S1P reduces HSR-induced microvascular leakage by preserving endothelial cell junctional structure and the endothelial glycocalyx through the protection of mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Junctional integrity in the mesenteric microcirculation was assessed by confocal microscopy. Cultured rat intestinal microvascular endothelial cells monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that in vivo, S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. S1P also protects against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III. Taken together, our data indicate that S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to the prevention of excess microvascular permeability.