Probiotics are defined as microorganisms that can exert health benefits for the host. Among the recognized probiotics, Bifidobacterium are the most frequently used probiotics in humans. The aim of this study was to evaluate the antidiabetic activity of Bifidobacterium strains isolated from breastfed infant faeces, both in vitro, using the Caco-2 monolayer transwell model, and in vivo, using a mice model of impaired glucose tolerance induced by a high-fat diet (HFD). The cell-free supernatant of Bifidobacterium lactis A12 showed better inhibitory activity of α-glucosidase and inhibited the glucose absorption and transport than B. lactis BB12, which is a typical probiotic with antidiabetic capabilities. B. lactis A12 improved the impaired glucose intolerance, restored islet function and morphology with insulin resistance induced by the HFD in C57BL/6J mice. Furthermore, in small intestine tissues, the cell-free supernatant of B. lactis A12 decreased the messenger RNA expressions of sucrase-isomaltase, live B. lactis A12 cells decreased glucose transporters 2. B. lactis A12 significantly stimulated the glucagon like peptide-1 (GLP-1) secretion and upregulated proglucagon messenger RNA levels. B. lactis A12 protect against the deleterious effects of HFD-induced diabetes by inhibiting the utilization, absorption, and transport of glucose by intestinal epithelial cells and promoting the expression and secretion of GLP-1. © 2020 Society of Chemical Industry.