Cathepsins are mammalian papain-like cysteine proteases that play an important role in numerous physiological and pathological processes. In the present study, various molecular dynamics (MD) simulations of pro- and mature human cathepsins L and O were performed. This study is the first to report MD simulations to complement the initial model structure of (pro-)cathepsin O through conformational sampling, thus offering insight into the maturation of procathepsin O, which to date has not been described experimentally. The overall fold of (pro-)cathepsin O appears very similar to that of (pro-)cathepsin L. The propeptide binding loop (PBL)-propeptide interface of both procathepsins is found to form a stable two-stranded β-sheet. Additional stabilization of the PBL-propeptide interface is provided by hydrophobic side chain contacts in procathepsin L, whereas this seems to be due to charge-dipole interactions in procathepsin O. Introduction of two mutations (L147P and G148P) into procathepsin O entails a significant loss of hydrogen bonding, disabling formation of the interfacial β-sheet. Simulations at different protonation states suggest that procathepsin L is more sensitive to a change in pH than procathepsin O. Potential differences between the maturation of procathepsin O and procathepsin L inferred from the MD simulations might be caused by (i) stronger PBL-propeptide interactions in procathepsin O due to salt-bridge formation across the interface, (ii) more limited entropic gain of the propeptide of procathepsin O upon release into the bulk solvent due to diverse conformational states sampled in the bound state, (iii) more pronounced entropic loss of the PBL in procathepsin O upon substrate binding caused by diverse conformational states sampled in the free, mature enzyme, and (iv) lower sensitivity of procathepsin O to pH change caused by the presence of fewer carboxylate groups at the PBL-propeptide interface.