Endothelial progenitor cells (EPC) significantly contribute to neovascularization and endothelial regeneration. Risk factors for coronary artery disease, particularly diabetes mellitus, reduce the number and functional activity of EPC. As we have recently shown, expression and activity of the matrix degrading cysteine protease cathepsin L in EPC is required for tissue invasion and EPC-mediated improvement of neovascularization. Therefore, we investigated the effect of high glucose and diabetes mellitus on EPC invasion and cathepsin L activity. Incubation of EPC with high levels of glucose (10–30 mM) dose-dependently decreased cathepsin L activity (glucose 20 mM: 67±4% compared to control; p<0.05) and protein expression (48±5% of control, p<0.05). In contrast, other proteases of the cathepsin family such as cathepsins D and O, and the matrix metalloproteinases MMP-2 and MMP-9 were not altered with high glucose. Cathepsin L mRNA was not affected suggesting that a posttranscriptional mechanism is responsible for cathepsin L down-regulation. As a functional consequence, high glucose significantly reduced the gelatinolytic activity and invasion of EPC (50±5% of control). Importantly, EPC of patients with type 2 diabetes revealed profoundly decreased cathepsin L expression and activity as compared to EPC derived from healthy controls. Taken together, high glucose significantly reduces the protein expression and activity of cathepsin L, which is involved in matrix degradation and required for invasion of EPC into the ischemic tissue, and, thereby, may limit the functional capacity of EPC to improve neovascularization in diabetics.