Abstract: To further understand the mechanism of Tamarix hispida V-ATPase under stresses, in the current study， differential centrifugation and discontinuous sucrose density gradient centrifugation were used to isolate and purify the T.hispida tonoplast. The results of Western blotting showed that the ThVHAc1 protein was screened, indicating that the isolated tonoplast contained V-ATPase. Furthermore, the sensitivity of tonoplast ATPase was determined by added with inhibitors NaNO3, Na3VO3, NaN3, and V-ATPase hydrolytic activity and overturn percentage were also determined. The results showed the tonoplast was mainly distributed on the 0-25% sucrose interface, and grinding for insolating the tonoplast showed high V-ATPase hydrolytic and percentage of overturn, indicating liquid nitrogen grinding combined with differential centrifugation and discontinuous density gradient centrifugation methods was effective to isolate T.hispida tonoplast.