Description: Cathepsin K (CatK) is one of the most potent mammalian collagenases. We showed previously the increased expression of CatK in human and animal atherosclerotic lesions. Here, we hypothesized that ablation of CatK mitigates injury-induced neointimal hyperplasia. Male wild-type (CatK +/+ ) and CatK-deficient (CatK –/– ) mice underwent ligation or a combination of ligation and polyethylene cuff-replacement injuries to the right common carotid artery just proximal to its bifurcation, and they were then processed for morphological and biochemical studies at specific time points. On operative day 28, CatK –/– significantly reduced neointimal formation and neovessel formation in both single- and combination-injured arteries compared with the Cat K +/+ mice. At early time points, CatK –/– reduced the lesion macrophage contents and medial smooth muscle cell proliferation, the mRNA levels of monocyte chemoattractant protein-1, toll-like receptor-2, toll-like receptor-4, chemokine ligand-12, and the gelatinolytic activity related to matrix metalloproteinase-2/-9. An aorta-explant assay revealed that smooth muscle cell movement was impaired in the CatK –/– mice compared with the CatK +/+ mice. In addition, the smooth muscle cells and macrophages from CatK –/– mice had less invasive ability through a reconstituted basement membrane barrier. This vasculoprotective effect was mimicked by Cat inhibition with trans -epoxysuccinyl-L-leucylamido-{4-guanidino} butane (E64 d ). These results demonstrate an essential role of CatK in neointimal lesion formation in response to injury, possibly via the reduction of toll-like receptor-2/-4–mediated inflammation and smooth muscle cell proliferation, suggesting a novel therapeutic strategy for the control of endovascular treatment–related restenosis by regulating CatK activity.