Combined quantum mechanical (QM) and molecular mechanical (MM) calculations are reported for the triosephosphate isomerase-catalyzed conversion of dihydroxy acetone phosphate into glyceraldehyde 3-phosphate. The minima and transition states for the relevant proton-transfer reactions have been located on QM/MM potential surfaces. The primary objective of this work is to study the sensitivity of optimized structures and relative energies toward variations in the QM/MM model, including the choice of the QM method, the size of the QM region, the size of the optimized MM region, and the treatment of the QM/MM boundary. The QM methods that have been applied in combination with the CHARMm force field range from semiempirical (AM1) to density functional (BP86, B3LYP) and ab initio (MP2) methods, the most extensive QM calculations involving 275 atoms and 2162 basis functions at the density functional level, Implications of the different choices of QM/MM options on the energy profile are discussed. From a mechanistic point of view, the present QM/MM results support a four-step proton-transfer pathway via an enediol, with involvement of neutral His95 acting as a proton donor, since the alternative direct intramolecular proton transfer in the enediolate is disfavored by the protein environment.