Salinity is one of the major environmental factors limiting plant growth and productivity. An H+-PPase gene, TsVP from Thellungiella halophila, was transferred into cotton (Gossypium hirsutum) in sense and antisense orientations under control of the cauliflower mosaic virus (CaMV) 35S promoter. Southern and Northern blotting analysis showed that the sense or antisense TsVP were integrated into the cotton genome and expressed. Transgenic plants overexpressing the vacuolar H+-PPase were much more resistant to 150 and 250 mM NaCl than the isogenic wild-type plants. In contrast, the plants from the antisense line (L2), with lower H+-PPase activity, were more sensitive to salinity than the wild-type plants. Overexpressing TsVP in cotton improved shoot and root growth and photosynthetic performance. These transgenic plants accumulated more Na+, K+, Ca2+, Cl- and soluble sugars in their root and leaf tissues under salinity conditions compared with the wild-type plants. The lower membrane ion leakage and malondialdehyde (MDA) level in these transgenic plants suggest that overexpression of H+-PPase causes the accumulation of Na+ and Cl- in vacuoles instead of in the cytoplasm, thus reducing their toxic effects. On the other hand, the increased accumulation of ions and sugars decreases the solute potential in cells, and facilitates water uptake under salinity, which is an important mechanism for the increased salt tolerance in TsVP-overexpressing cotton.