In plants, hexokinase (HXK, EC 2.7.1.1), an enzyme normally involved in hexose phosphorylation, plays an important role in sugar sensing and signaling. The hexokinase activity of grape HXKs was confirmed by functional complementation of the hexokinase-deficient yeast strain YSH7.4-3C (hxk1, hxk2, glk1). HXK1 and HXK2 were able to complement this mutant. The subcellular localization of HXK1 and HXK2, observed with green fluorescent protein fusion constructs, indicated that HXK1 localized to the cytosol while HXK2 was a nuclear-targeted hexokinase. Gibberellin (GA3) control various processes across plant life and has been involved in sugar accumulation. The coordinated regulation of exogenous GA3 with Glc on CWINV, SuSy1, or SuSy2 expressions indicated that GA3 can relieve the repression of Glc on CWINV or SuSy1 expression, and the repression of GA3 on SuSy2 expression overrides the Glc-inductive effect, resulting in the down-regulation of SuSy2 expression. It was concluded that GA3 negatively interfere with Glc signal transduction depending on hexokinase phosphorylation. GA3 might regulate CWINV, SuSy1 or SuSy2 expression to in order to maintain an intracellular sugar levels and normal cell metabolism. Our results provide new insights into the crosstalk mechanism of GA3 and Glc signaling depending on hexokinase in grape berry sugar accumulation.