Cathepsin K has been shown to exhibit antimicrobial and anti-inflammatory activities in the mouse colon. To further elucidate its role, we used Ctsk-/- mice and demonstrated that the absence of cathepsin K was accompanied by elevated protein levels of related cysteine cathepsins (cathepsins B, L, and X) in the colon. In principle, such changes could result in altered subcellular localization; however, the trafficking of cysteine cathepsins was not affected in the colon of Ctsk-/- mice. However, cathepsin K deficiency affected the extracellular matrix constituents, as higher amounts of collagen IV and laminin were observed. Moreover, the localization pattern of the intercellular junction proteins E-cadherin and occludin was altered in the colon of Ctsk-/- mice, suggesting potential impairment of the barrier function. Thus, we used an ex vivo method for assessing the mucus layers and showed that the absence of cathepsin K had no influence on mucus organization and growth. The data of this study support the notion that cathepsin K contributes to intestinal homeostasis and tissue architecture, but the lack of cathepsin K activity is not expected to affect the mucus-depending barrier functions of the mouse colon. These results are important with regard to oral administration of cathepsin K inhibitors that are currently under investigation in clinical trials.