The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions. This is relevant for the genetic engineering of crops with economic interest that do not naturally possess efficient pathways for the synthesis of glycine betaine and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, the best characterized choline-oxidizing enzyme is the flavin-dependent choline oxidase from Arthrobacter globiformis, for which structural, mechanistic, and biochemical data are available. Here, we have replaced a hydrophobic residue (Val464) lining the active site cavity close to the N(5) atom of the flavin with threonine or alanine to investigate its role in the reaction of choline oxidation catalyzed by choline oxidase. The reductive half-reactions of the enzyme variants containing Thr464 or Ala464 were investigated using substrate and solvent kinetic isotope effects, solvent viscosity effects, and proton inventories. Replacement of Val464 with threonine or alanine uncovered a kinetically slow equilibrium between a catalytically incompetent form of enzyme and an active species that can efficiently oxidize choline. In both variants, the active form of enzyme shows a decreased rate of hydroxyl proton abstraction from the alcohol substrate, with minimal changes in the subsequent rate of hydride ion transfer to the flavin. This study therefore establishes that a hydrophobic residue not directly participating in catalysis plays important roles in the reaction of choline oxidation catalyzed by choline oxidase.