The survival of plant cells strongly depends on maintaining or adjusting the activity of the V-ATPase under drought conditions. In this study, a V-ATPase gene, VHA-B, was identified in an apple cDNA library, and an analysis of the deduced MdVHA-B protein sequence showed that it clusters with VHA-B proteins of other species. The expression of MdVHA-B was induced by abiotic stress, including abscisic acid (ABA), salinity, drought and low and high temperatures. Furthermore, the over-expression of MdVHA-B in transgenic tomato plants resulted in a high tolerance to drought stress, reduced relative water loss and malondialdehyde contents and increased levels of free proline and H + -ATPase activity compared to the wild-type plants (WT). Additionally, it has similar sensibility to exogenous ABA in transgenic MdVHA-B tomato seeds and seedlings, while more sensitive to polyethylene glycol in transgenic tomato compared with WT. This study indicates that MdVHA-B plays an important role in drought stress, yet may act independently of the ABA signaling pathway.