In this paper, we have studied the catalytic mechanism of L-asparaginase II computationally. The reaction mechanism was investigated using the ONIOM methodology. For the geometry optimization we used the B3LYP/6-31G(d):AM1 level of theory, and for the single points we used the M06-2X/6-311+ +G(2d,2p):M06-2X/6-31G(d) level of theory. It was demonstrated that the full mechanism involves three sequential steps and requires the nucleophilic attack of a water molecule on the substrate prior to the release of ammonia. There are three rate-limiting states, which are the reactants, the first transition state, and the last transition state. The energetic span is 20.2 kcal/mol, which is consistent with the experimental value of 16 kcal/mol. The full reaction is almost thermoneutral. The proposed catalytic mechanism involves two catalytic triads that play different roles in the reaction. The first triad, Thr12-Lys162-Asp90, acts by deprotonating a water molecule that subsequently binds to the substrate. The second triad, Thr12-Ty25-Glu283, acts by stabilizing the tetrahedral intermediate that is formed after the nucleophilic attack of the water molecule to the substrate. We have shown that a well-known Thr12-substrate covalent intermediate is not formed in the wild-type mechanism, even though our results suggest that its formation is expected in the Thr89Val mutant. These results have provided a new understanding of the catalytic mechanism of L-asparaginases that is in agreement with the available experimental data, even though it is different from all earlier proposals. This is of particular importance since this enzyme is currently used as a chemotherapeutic drug against several types of cancer and in the food industry to control the levels of acrylamide in food.