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Sugar content directly affects grape (Vitis vinifera L.) berry quality and the resulting wine. Therefore, it is of great importance to study and explore novel genes that affect sugar accumulation in grapes. Glycosyl hydrolases (GHs) are key enzymes hydrolyzing polysaccharides into monosaccharides and play important roles in the regulation of carbohydrate metabolism. Nevertheless, the impact of GHs on the regulation of sugar accumulation in plants has rarely been investigated. In this study, we identified 11 putative GH genes in grapevines by phylogeny analysis. RNA-seq and quantitative real-time PCR results demonstrated that the expression level of VvGH9 was higher during the fruit set stage, which had lower sugar content than the véraison and ripe stages. Treatment of grape berries with exogenous sugar two weeks before véraison revealed that VvGH9 was rapidly induced by sucrose, fructose, and glucose. When ‘41B’ calli was treated with different concentrations of glucose, VvGH9 expression increased at first and then decreased with the increase of glucose concentration. Overexpression of VvGH9 in grape calli and tomatoes also confirmed that this gene could contribute to sugar accumulation. All the above results demonstrated that VvGH9 promotes sugar accumulation under low sugar content in plants.