β-glucosidases are enzymes that hydrolyze β-1,4-glycosidic bonds to release non-reducing terminal glucosyl residues from glycosides and oligosaccharides, thus have significant application potential in industries.However, most β-glucosidases are feedback inhibited by the glucose product, which restricts their application.Remarkably,some β-glucosidases belonging to the GH (glycoside hydrolase) 1 family are tolerant to or even stimulated by glucose.Elucidation of the mechanisms of glucose tolerance and stimulation of the GH1 β-glucosidases will be crucial to improve their application through enzyme engineering.In this study, by comparing the primary and tertiary structures of two GH1 β-glucosidases with distinct glucose dependence, some putative glucose-dependence relevant sites were selected to be mutated to investigate their exact roles.Characterization, including biochemical and structural, of the mutants suggested that some sites at the entrance and middle of the substrate channel regulate the effects of glucose, and the relative affinity/preference of these sites binding to glucose modulates the glucose dependence.A mechanism was therefore proposed to interpret the glucose effects on GH1 β-glucosidases.Our research improves and deepens understanding of the properties of GH1 β-glycosidases and may be reference for other enzymes particularly in terms of product effects on activity.This work was supported by the Chinese National Natural Science Foundation, grant number 31300062.