Mature pear fruit lose water rapidly, and the later the harvest, the more the water loss, severely limiting the fruit quality and economic benefits of pears. However, the molecular mechanism of pear fruit water loss is complicated and still far from being understood. In this study, the flesh of juice pear fruit (JP) and water loss pear fruit (WP) were used for proteomic analyses. Compared WP with JP, we obtained 159 differentially expressed proteins, especially cell wall metabolic and endomembrane transport proteins. Moreover, the cell membrane permeability and polygalacturonase (PG) activity were increased in WP compared with JP, illustrating that water loss of pear fruit may be related to the composition and metabolism of the cell wall. Furthermore, aquaporin coding genes AQPs and the cell wall metabolic gene PbPG were cloned, and sequence analysis indicated their important roles in fruit water loss. We then verified the function of PbPG in fruit water loss and further suggested that PbPG accelerated water loss of pear fruit was achieved by degrading cell wall polysaccharides, increasing intercellular layer and AQP-encoding gene expression. These findings will provide novel insights into the molecular mechanisms behind pear fruit water loss.