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MPO (myeloperoxidase) is a peroxidase enzyme secreted by activated leukocytes that plays a pathogenic role in cardiovascular disease, mainly by initiating endothelial dysfunction. The molecular mechanisms of the endothelial damaging action of MPO remain though largely elusive. Calpain is a calcium-dependent protease expressed in the vascular wall. Activation of calpains has been implicated in inflammatory disorders of the vasculature. Using endothelial cells and genetically modified mice, this study identifies the µ-calpain isoform as novel downstream signaling target of MPO in endothelial dysfunction. Mouse lung microvascular endothelial cells were stimulated with 10 nmol/L MPO for 180 minutes. MPO denitrosylated µ-calpain C-terminus domain, and time dependently activated µ-calpain, but not the m-calpain isoform. MPO also reduced Thr172 AMPK (AMP-activated protein kinase) and Ser1177 eNOS (endothelial nitric oxide synthase) phosphorylation via upregulation of PP2A (protein phosphatase 2) expression. At the functional level, MPO increased endothelial VCAM-1 (vascular cell adhesion molecule 1) abundance and the adhesion of leukocytes to the mouse aorta. In MPO-treated endothelial cells, pharmacological inhibition of calpain activity attenuated expression of VCAM-1 and PP2A, and restored Thr172 AMPK and Ser1177 eNOS phosphorylation. Compared with wild-type mice, µ-calpain deficient mice experienced reduced leukocyte adhesion to the aortic endothelium in response to MPO. Our data first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO. The MPO/calpain/PP2A signaling pathway may provide novel pharmacological targets for the treatment of inflammatory vascular disorders.