The NADPH-dependent L-sorbose reductase (SR) of L-sorbose-producing Gluconobacter suboxydans IFO 3291 contributes to intracellular L-sorbose assimilation. The gene disruptant showed no SR activity and did not assimilate the once-produced L-sorbose, indicating that the SR functions mainly as an L-sorbose-reducing enzyme in vivo and not as a D-sorbitol-oxidizing enzyme.