The vacuole is by far the largest multifunctional organelle in fruits and plays a functional role in fruit development and fruit quality. Despite its significance, little information exists pertaining to the role of the vacuolar membrane (tonoplast) in the process of fruit senescence. In the present study, an iTRAQ-based quantitative proteomic approach was used to characterize the dynamic alterations in the tonoplast proteome during fruit senescence. Tonoplasts were purified from apple fruit at various stages of senescence using an iodixanol step gradient protocol. A total of 345 tonoplast-related proteins were identified with diverse functions such as transporters and proton pumps, signal transduction, membrane fusion or vesicle trafficking, cellular metabolic process, defense response, protein folding and degradation, and cytoskeleton. Changes in protein abundance during storage were characterized for the identified proteins. A total of 22 proteins displayed differential levels of abundance during storage. The senescence-related tonoplast proteins mostly function in the transportation of metabolites, signal transduction, membrane trafficking, and stress response. RT-qPCR analysis was used to quantify the level of expression of nine genes encoding some of the differentially abundant proteins. The results of this study provide new information regarding the function of the tonoplast during fruit senescence.