Human cathepsin K (hCatK), which is highly expressed in osteoclasts, has the noteworthy ability to cleave type I and II collagens in their helical domain. Its collagenase potency depends strictly on the formation of an oligomeric complex with chondroitin 4-sulfate (C4-S). Accordingly, hCatK is a pivotal protease involved in bone resorption and is an attractive target for the treatment of osteoporosis. As rat is a common animal model for the evaluation of hCatK inhibitors, we conducted a comparative analysis of rat CatK (rCatK) and hCatK, which share a high degree of identity (88%) and similarity (93%). The pH activity profile of both enzymes displayed a similar bell-shaped curve (optimal pH: 6.4). Presence of Ser134 and Val160 in the S2 pocket of rCatK instead of Ala and Leu residues, respectively, in hCatK, led to a weaker peptidase activity, as observed for mouse CatK. Also, regardless of the presence of C4-S, rCatK cleaved in the nonhelical telopeptide regions of both type I (tail) and type II (articular joint) rat collagens. Structure-based computational analyses (electrostatic potential, molecular docking, molecular dynamics, free energy calculations) sustained that the C4-S mediated collagenolytic activity of rCatK obeys distinct molecular interactions from those of hCatK. Additionally, T-kininogen (a.k.a. thiostatin), a unique rat serum acute phase molecule, acted as a tight-binding inhibitor of hCatK (Ki = 0.11 ± 0.05 nM). Taken into account the increase of T-Kininogen level in inflamed rat sera, this may raise the question of the appropriateness to evaluate pharmacological hCatK inhibitors in this peculiar animal model.