Cyclosporine, a widely used immunosuppressant in organ transplantation and in treatment of various autoimmune diseases, activates the unfolded protein response (UPR), an ER stress coping response. In this study we discovered a new and unanticipated cyclosporine-dependent signaling pathway, with cyclosporine triggering direct activation of the UPR. COX-2 binds to and activates IRE1Î±, leading to IRE1Î± splicing of XBP1 mRNA. Molecular interaction and modeling analyses identified a novel interaction site for cyclosporine with COX-2 which caused enhancement of COX-2 enzymatic activity required for activation of the IRE1Î± branch of the UPR. Cyclosporine-dependent activation of COX-2 and IRE1Î± in mice indicated that cyclosporine-COX-2-IRE1Î± signaling pathway was functional in vivo. These findings identify COX-2 as a new IRE1Î± binding partner and regulator of the IRE1Î± branch of the UPR pathway, and establishes the mechanism underlying cytotoxicity associated with chronic cyclosporine exposure.