Fruit abscission is a severe hindrance to commercial crop production, and a lack of carbohydrates causes fruit abscission to intensify in a variety of plant species. However, the precise mechanism by which carbohydrates affect fruit setting potential has yet to be determined. In the current study, we noticed negative correlation between hexose level and fruit setting by comparing different cultivars, bearing shoots of varying diameters, and girdling and defoliation treatments. And the cumulative fruit dropping rate was significantly reduced in response to exogenous glucose dipping. These results suggested that hexose, especially glucose, is the key player in lowering litchi fruit abscission. Moreover, five putative litchi hexokinase genes (LcHXKs) were isolated and the subcellular localization as well as activity of their expressed proteins in catalyzing hexose phosphorylation were investigated. LcHXK2 was only found in mitochondria and expressed catalytic protein, whereas the other four HXKs were found in both mitochondria and nuclei and had no activity in catalyzing hexose phosphorylation. LcHXK1 and LcHXK4 were found in the same cluster as previously reported hexose sensors AtHXK1 and MdHXK1. Furthermore, VIGS-mediated silencing assay confirms that LcHXK1 suppression increases fruit abscission. These findings revealed that LcHXK1 functions as hexose sensor, negatively regulating litchi fruit abscission.