We have previously shown that vacuolar H+-ATPase subcomplex Vo from mung bean contains subunit d, however, its sequence and function were unknown. In the present study, we report the cloning and recombinant over expression of subunit d from mung bean in E. coli. To study the function of subunit d, two vacuolar H+-ATPase subcomplexes Vo from mung bean were purified-one containing subunits a and c(c′,c″) and the other containing subunits a, c(c′,c″) and d. After reconstitution of the purified Vo subcomplexes into liposomes, the proton translocation was studied. Our results show that the Vo subcomplex in the absence of subunit d is a passive proton channel, while the Vo subcomplex in the presence of the subunit d is not. Taken together, our data supports the conclusion that the subunit d of the plant vacuolar H+-ATPase from mung bean is positioned at the central stalk and involved in the proton translocation across the tonoplast membrane.