G protein–coupled receptors (GPCRs) of endothelial cells transmit diverse intracellular signals that regulate adherens junction (AJ) permeability. Increased endothelial permeability contributes to pathological processes such as inflammation, atherogenesis, and acute lung injury. Thrombin, a serine protease, and sphingosine-1-phosphate (S1P), a bioactive lipid, regulate endothelial barrier function by activating their respective GPCRs—the protease-activated receptor PAR₁ and the S1P receptor S1P₁—which initiate intracellular signals that regulate AJ integrity and cytoskeleton organization. The distinct patterns of PAR₁ and S1P₁ signal transduction underlie the functional antagonism between thrombin and S1P. Evidence points to a role for activation of the S1P₁ receptor that is induced by PAR₁-mediated signaling in the mechanism of AJ reannealing and endothelial barrier repair. Understanding the molecular basis of AJ integrity in the context of inflammation is important in developing novel anti-inflammatory therapeutics. This Review provides a working model for molecular mechanisms for the dual regulation of endothelial barrier function.