Background: Prostaglandin I₂, or PGI₂, has been shown to attenuate vascular constriction, hyperpermeability, inflammation, and acute lung injury. However, molecular mechanisms of PGI₂ protective effects on pulmonary endothelial cells (EC) are not well understood. We tested a role of cAMP-activated Epac-Rap1 pathway in the barrier protective effects of PGI₂ analogue iloprost in the murine model of ventilator-induced lung injury. Methods: Mice were treated with iloprost (2 μg/kg) after onset of high tidal volume ventilation (HTV, 30 ml/kg, 4 hrs). Bronchoalveolar lavage (BAL), histological analysis, and measurements of Evans blue accumulation were performed. In vitro, microvascular EC barrier function was assessed by morphological analysis of agonist-induced gap formation, and monitoring of Rho pathway activation and EC permeability. Results: Iloprost reduced BAL protein content, neutrophil accumulation, capillary filtration coefficient, and Evans blue albumin extravasation caused by HTV. SiRNA-based Rap1 knockdown inhibited protective effects of iloprost. In vitro, iloprost increased barrier properties of lung microvascular endothelium and alleviated thrombin-induced EC barrier disruption. In line with in vivo results, Rap1 depletion attenuated protective effects of iloprost in the thrombin model of EC permeability. Conclusion: These data describe for the first time protective effects for Rap1-dependent signaling against ventilator induced lung injury and pulmonary endothelial barrier dysfunction.