L-Rhamnose isomerase (L-RhI) has been found in many microorganisms and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Interestingly, Pseudomonas stutzeri L-RhI (P. stutzeri L-RhI) exhibits a much broader substrate specificity than others such as Escherichia coli L-RhI (E. coli L-RhI) and catalyzes the interconversion of many aldoses and ketoses. To elucidate the uniqueness of P. stutzeri L-RhI and the mechanism of enzymatic catalysis, we performed dual-level combined QM/MM molecular dynamics simulations on P. stutzeri L-RhI with a number of substrates. Calculations show that the reversible process between aldoses and ketoses can be rationalized by a zwitterion intermediate mechanism that involves both proton and hydride transfers. Predicted free energy barriers in the rate-determining. step are 8.9 kcal/mol for L-rhamnose and 13.6 kcal/mol for D-allose, respectively, in good agreement with the experimental characterization of relative substrate reactivity. Conformational and hydrogen bond analyses of the active domain and evaluation of electrostatic and van der Waals (vdW) interactions between substrates and surrounding residues provide a basis to understand the catalytic role of conserved residues, the substrate specificity, and the relative activity of favorable substrates in P. stutzeri L-RhI.