Activated protein C (APC) has potent anticoagulant and anti-inflammatory properties that limit clot formation, inhibit apoptosis, and protect vascular endothelial cell barrier integrity. In this study, the role of N-linked glycans in modulating APC endothelial cytoprotective signaling via endothelial cell protein C receptor/protease-activated receptor 1 (PAR1) was investigated. Enzymatic digestion of APC N-linked glycans (PNG-APC) decreased the APC concentration required to achieve half-maximal inhibition of thrombin-induced endothelial cell barrier permeability by 6-fold. Furthermore, PNG-APC exhibited increased protection against staurosporine-induced endothelial cell apoptosis when compared with untreated APC. To investigate the specific N-linked glycans responsible, recombinant APC variants were generated in which each N-linked glycan attachment site was eliminated. Of these, APC-N329Q was up to 5-fold more efficient in protecting endothelial barrier function when compared with wild type APC. Based on these findings, an APC variant (APC-L38D/N329Q) was generated with minimal anticoagulant activity, but 5-fold enhanced endothelial barrier protective function and 30-fold improved anti-apoptotic function when compared with wild type APC. These data highlight the previously unidentified role of APC N-linked glycosylation in modulating endothelial cell protein C receptor-dependent cytoprotective signaling via PAR1. Furthermore, our data suggest that plasma β-protein C, characterized by aberrant N-linked glycosylation at Asn-329, may be particularly important for maintenance of APC cytoprotective functions in vivo.