Although zinc is an essential micronutrient for all living organisms, zinc is harmful to cells at high levels. In the presence of excess zinc, plants exhibit several major symptoms, including root growth inhibition, abnormal root hair morphology and chlorosis. To dissect the molecular mechanisms underlying the effects of excess zinc on plant cells, we used aniTRA Q-based quantitative proteomics approach to analyze the microsomal protein profiles of Arabidopsis roots from wild-type (WT) plants and de-etiolated 3-1 (det3-1), a vacuolar H+ -AT Pase (V-AT Pase) subunit C-defective mutant. A comparative analysis of the iTRA Q data from WT and det3-1 plants exposed to excess zinc suggests that the reduction in V-AT Pase subunit levels and its activity are the cause of the symptoms of zinc toxicity, including the inhibition of cell expansion. Provided that reduced V-AT Pase activity in the trans-Golgi network (TGN) alone can inhibit cell expansion, it is possible that the det3-1 mutant phenotype is caused mainly by a defect in TGN acidification, leading to reduced cell wall component trafficking and cell expansion in the presence of excess zinc. To evaluate the contribution of V-AT Pase activity to vacuolar acidification under excess zinc, the vacuolar pH was measured. Our results indicate clear alkalinization of deep cell vacuoles treated with 300 μM ZnSO4.