A kinetic study of the regeneration reaction of vitamin E by ubiquinol was carried out by means of double-mixing stopped-flow spectroscopy. A substantial deuterium kinetic-isotope effect was observed on the second-order rate constant and the activation energy. In the regeneration reaction of alpha-tocopherol, deuteration of ubiquinol increased and decreased the activation energy and the second-order rate constant by 6.1 kJ/mol and a factor of 18.3, respectively. From this result, it is considered that proton tunneling plays an important role in the regeneration reaction of vitamin E by ubiquinol. The conditions under which the tunneling effect becomes an important factor were discussed in conjunction of our experimental results.