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Background: Similar to the high-fat diet (HFD), the high-glycemic diet (HGD) contributes to the development and progression of type 2 diabetes mellitus (T2DM). However, the effect of HGD on gastrointestinal motility in T2DM and its u nderlying mechanisms remain unclear. Methods: Thirty C57BL/6J mice were randomly designated into the normal-feeding diet (NFD) group, HFD group, and HGD group. The plasma glucose, plasma insulin, and gastrointestinal motility were examined. Meanwhile, the tension of isolated colonic smooth muscle rings was calculated, and the gut microbiota was analyzed by 16s rDNA high-throughput sequencing. Result: After 16 weeks of HGD feeding, obesity, hyperglycemia, insulin resistance, and constipation were observed in HGD mice. Autonomic contraction frequency of the colonic neuromuscular system and electrical field stimulation-induced contractions were reduced in HGD mice. On the contrary, neuronal nitric oxide synthase activity and neuromuscular relaxation were found to be enhanced. Finally, gut microbiota analysis revealed that Rhodospirillaceae abundance significantly increased at the family level in HGD mice. At the genus level, the abundance of Insolitispirillum increased remarkably, whereas Turicibacter abundance decreased significantly in HGD mice. Conclusion: HGD induced constipation in obese diabetic mice, which we speculated that it may be related to neuromuscular dysmotility and intestinal microbiota dysbiosis.