Pot-pollen is a mixture of pollen and nectar from flowers combined with salivary substances of stingless bees, which together are fermented inside cerumen pots. As pot-pollen is rich in polyphenols, we hypothesized that dietary ingestion could modulate obesity, glucose metabolism, and the gut microbiota in an animal model of diet-induced obesity. Male C57BL/6J mice were fed a low-fat/low-sucrose diet (LF/LS), a HF/HS diet or a HF/HS diet containing 0.1% pot-pollen (HF/HS-PP) for 12 weeks. In HF/HS-fed mice, pot-pollen supplementation decreased fasting blood glucose and increased glucose-stimulated insulin secretion without modifying weight gain, body composition, glucose tolerance, and insulin sensitivity. Intake of pot-pollen resulted in changes of the gut microbiota, including a decrease in the abundance of the Rikenellaceae RC9 gut group and Lactobacillus, and an increase in the abundance of Romboutsia. Correlations between genus abundances and metabolic changes in response to supplementation indicated that the gut microbiota contributed to the positive effects of pot-pollen ingestion on fasting glucose. Pot-pollen supplementation-associated changes in the gut microbiota composition correlated with the lowering of fasting glucose levels without modulating weight gain.