Glucokinase (GK) is a glycolic enzyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate in the first step of glycolysis. Thus alteration in GK activity plays an important role in abnormal glycemia. A great deal of research about maturity-onset diabetes of the young 2 (MODY 2) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI) confirmed that the change of GK activity was associated with diabetes. The explicit molecular dynamics simulations and implicit solvent binding free-energy calculations were investigated to understand the activation mechanism of GK point mutation M197V (Met197 →Val). The root mean square fluctuation (RMSF) and dynamic cross-correlation matrices (DCCM) conformation analysis showed that GK M197V mutation resulted in a more stable active conformation. The binding free-energy analysis demonstrated that the GK M197V mutation increased its binding affinity with glucose. The difference of binding free-energy between wild-type (WT) GK and M197V GK was in well agreement with previous experimental results. Our results can interpret the active mechanism of GK M197V mutation from the atom level perfectly.