Endothelial cells normally form a dynamically regulated barrier at the blood-tissue interface, and breakdown of this barrier is a key pathogenic factor in inflammatory disorders such as sepsis. Pro-inflammatory signaling by the blood coagulation protease thrombin through protease activated receptor-1 (PAR1) can disrupt endothelial barrier integrity, whereas the bioactive lipid sphingosine 1-phosphate (S1P) recently has been demonstrated to have potent barrier protective effects. Activated protein C (APC) inhibits thrombin generation and has potent anti-inflammatory effects. Here, we show that APC enhanced endothelial barrier integrity in a dual-chamber system dependent on binding to endothelial protein C receptor, activation of PAR1, and activity of cellular sphingosine kinase. Small interfering RNA that targets sphingosine kinase-1 or S1P receptor-1 blocked this protective signaling by APC. Incubation of cells with PAR1 agonist peptide or low concentrations of thrombin (approximately 40 pM) had a similar barrier-enhancing effect. These results demonstrate that PAR1 activation on endothelial cells can have opposite biologic effects, reveal a role for cross-communication between the prototypical barrier-protective S1P and barrier-disruptive PAR1 pathway, and suggest that S1P receptor-1 mediates protective effects of APC in systemic inflammation.