(KARI) catalyzes the conversion of (S)-2-acetolactate or (S)-2-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate, the second step in the biosynthesis of branched chain amino acids (BCAAs). Because the BCAA biosynthetic pathway is present in bacteria, plants, and fungi, but absent in animals, it is an excellent target for the development of new-generation antibiotics and herbicides. Nevertheless, the mechanism of the KARI-catalyzed reaction has not yet been fully solved. In this study, we used iterative molecular dynamics (MD) flexible fitting–Rosetta techniques to optimize the three-dimensional solution structure of archaea KARI from Sulfolobus solfataricus (Sso-KARI) determined from cryo-electron microscopy. On the basis of the structure of the Sso-KARI/2Mg2+/NADH/(S)-2-acetolactate complex, we deciphered the catalytic mechanism of the KARI-mediated reaction through hybrid quantum mechanics/molecular mechanics MD simulations in conjunction with umbrella sampling. With an activation energy of only 6.06 kcal/mol, a water-mediated, metal-catalyzed, base-induced (WMMCBI) mechanism was preferred for deprotonation of the tertiary OH group of (S)-2-acetolactate in Sso-KARI. The WMMCBI mechanism for double proton transfer occurred within a proton wire route with two steps involving the formation of hydroxide: (i) Glu233 served as a general base to deprotonate the Mg2+-bound water, forming a hydroxide-coordinated Mg2+ ion; (ii) this hydroxide ion acted as a strong base that rapidly deprotonated the ternary OH group of the substrate. In contrast, the direct deprotonation of the substrate by Glu233 was kinetically unfavorable. This mechanism suggests a novel approach for designing catalysts for deprotonation and provides clues for the development of new-generation antibiotics and herbicides.