Aquaporin is a water channel that increases water permeability through membranous structures. In plants, vacuoles are essential organelles that undergo dynamic volume changes during cell growth. To understand the contribution of aquaporins to plant cell growth, we developed a transgenic tobacco BY-2 cell line overexpressing the tonoplast intrinsic protein (TIP), gammaTIP. Vacuolar membranes of isolated vacuoles from gammaTIP-overexpressing cells showed higher water permeation activities than those from wild-type cells. We then examined the role of gammaTIP in vacuolar regeneration of evacuolated tobacco BY-2 protoplasts (miniprotoplasts). Vacuolar regeneration from thin to thick tube-network vacuoles and subsequent development of large vacuoles was accelerated in miniprotoplasts of this cell line. A parallel increase in the rate of cell expansion indicated a tight relationship between vacuolar development and cellular volume increases. Interestingly, overexpression of tobacco gammaTIP also enhanced cell division. Thus, increased vacuolar aquaporin activity may accelerate both cell expansion and cell division by increasing water permeability through the vacuolar membrane.