Proton pumps participate in several aspects of endocytic protein trafficking. However, their involvement specifically in the Glut4 pathway has been a matter of great controversy. Here, we report that incubation of 3T3-L1 adipocytes with specific inhibitors of V-type ATPase, Concanamycin A and Bafilomycin A1, inhibits insulin regulated glucose transport and results in accumulation of Glut4 in heavy rapidly sedimenting intracellular membranes. Correspondingly, the amount of small responsive Glut4-vesicles in Concanamycin A- and Bafilomycin A1-treated cells is decreased. We conclude that these drugs block translocation of Glut4 in adipose cells by inhibiting formation of small insulin-responsive vesicles on donor intracellular membranes. At the same time, proton pump inhibitors do not affect insulin-dependent translocation of preexisting vesicles or Glut4 sorting in recycling endosomes. On the contrary, Wortmannin acutely inhibits insulin-dependent translocation of the pre-existing vesicles but has no effect on vesicle formation.