Plant vacuoles play essential roles in many physiological processes, particularly in mineral nutrition, turgor provision and cellular signalling. The vacuolar membrane, the tonoplast, contains many membrane transporters that are critical in the execution of these processes. However, although increasing knowledge is available about the identity of proteins involved in these processes very little is known about the regulation of tonoplast transporters. By studying the phosphoproteome of tonoplast-enriched membranes, we identified 66 phosphorylation sites on 58 membrane proteins. Amongst these, 31 sites were identified in 28 membrane transporters of various families including tonoplast anion transporters of the CLC family, potassium transporters of the KUP family, tonoplast sugar transporters and ABC transporters. In a number of cases, the detected sites were well conserved across isoforms of one family pointing to common mechanisms of regulation. In other cases, isoform-unique sites were present, suggesting regulatory mechanisms tailored to the function of individual proteins. These results provide the basis for future studies to elucidate the mechanistic regulation of tonoplast membrane transporters.