Pteris vittata, the first known arsenic hyperaccumulating plant, can accumulate very high concentration arsenic in its aboveground tissues, while low in roots. Previous studies have suggested that arsenic vacuole compartmentalization may play an important role in the arsenic-hyperaccumulation in P. vittata, but the mechanism(s) of arsenic transport to vacuole are largely unknown. We obtained tonoplast isolated from fronds of P. vittata sporophyte grown under minus and 1 mM arsenate for 3 weeks by iodixanol step gradient centrifugation method, and then used TMPP protein labeling technology followed by liquid chromatography—a linear ion trap-Orbitrap hybrid mass spectrometer analysis for the quantitative detection of proteins. And we designed and used an “artificial” database for database searching. In total, 56 tonoplast proteins were identified; more than 70% of them were transport proteins. Under arsenate treatment, one TDT transporter protein, a member of the TerC family and a PDR-like protein were upregulated differentially. While V-ATPase subunits c, E, and G, and V-PPase, were downregulated. Additionally, the identified tonoplast proteins in our present study provide an informative basis for arsenic carriers or channels and help to clarify the regulation of tonoplast arsenic transport processes in P. vittata.