Objective— Activated protein C (APC), a plasma serine protease, initiates cell signaling that protects endothelial cells from apoptosis and endothelial barrier disruption. Apolipoprotein E receptor 2 (ApoER2; LRP8 ) is a receptor known for mediating signaling initiated by reelin in neurons. ApoER2 contributes to APC-initiated signaling in monocytic U937 cells. The objective was to determine whether ApoER2 is required for APC’s beneficial signaling in the endothelial cell surrogate EA.hy926 line. Approach and Results— We used small interfering RNA and inhibitors to probe requirements for specific receptors for APC’s antiapoptotic activity and for phosphorylation of disabled-1 by Src family kinases and of Akt. When small interfering RNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was used, APC’s antiapoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2- to 3-fold increased phosphorylation of Ser473-Akt and Tyr232-disabled-1, a phosphorylation known to trigger disabled-1–mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 small interfering RNA or by inhibitors of Src (PP2), phosphatidylinositol-3 kinase (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 small interfering RNA blocked the ability of APC to prevent thrombin-induced endothelial barrier disruption in TransEndothelial Resistance assays. Binding studies using purified APC and purified immobilized wild-type and mutated ApoER2 ectodomains suggested that APC binding involves Lys49, Asp50, and Trp64 on the surface of the N-terminal LA1 domain of ApoER2. Conclusions— ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial antiapoptotic and barrier protective signaling.