The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), increases the level or activity of several transcription factors that can induce the inducible nitric oxide synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by E. coli endotoxin (LPS) and interferon- (IFN) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS/IFN. There was no effect on induction of iNOS mRNA, suggesting a post-transcriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS/IFN was cytotoxic to MAEC lacking PIN1, but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester. Consistent with post-transcriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor, carbobenzoxy-valinyl-phenylalaninal, but not by the selective proteasome inhibitor, epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull-down of iNOS with a wildtype glutathione-S-transferase-PIN1 fusion protein, but not with a WW domain mutant of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.