ABSTRACT: BACKGROUND:The health of honeybee colonies is critical for bee products and agricultural production, and colony health is closely associated with the bacteria in the guts of honeybees. Although colony loss in winter is now the primary restriction in beekeeping, the effects of different sugars as winter food on the health of honeybee colonies are not well understood. Therefore, in this study, the influence of different sugar diets on honeybee gut bacteria during overwintering was examined. RESULTS:The bacterial communities in honeybee midguts and hindguts before winter and after bees were fed honey, sucrose, and high-fructose syrup as winter-food were determined by targeting the V3-V4 region of 16S rDNA using the Illumina MiSeq platform. The dominant microbiota in honeybee guts were the phyla Proteobacteria (63.17%), Firmicutes (17.61%; Lactobacillus, 15.91%), Actinobacteria (4.06%; Bifidobacterium, 3.34%), and Bacteroidetes (1.72%). The dominant taxa were conserved and not affected by season, type of overwintering sugar, or spatial position in the gut. However, the relative abundance of the dominant taxa was affected by those factors. In the midgut, microbial diversity of the sucrose group was higher than that of the honey and high-fructose syrup groups, but in the hindgut, microbial diversity of the honey and high-fructose groups was higher than that in the sucrose group. Sucrose increased the relative abundance of Actinobacteria (Bifidobacteriales Bifidobacteriaceae) and Alphaproteobacteria (Rhizobiales and Mitochondria) of honeybee midgut, and honey enriched the Bacteroidetes and Gammaproteobacteria (Pasteurellales) in honeybee hindgut. High-fructose syrup increased the relative abundance of Betaproteobacteria (Neisseriales: Neisseriaceae) of the midgut. CONCLUSION:The type of sugar used as winter food affected the relative abundance of the dominant bacterial communities in honeybee guts, not the taxa, which could affect the health and safety of honeybee colonies during overwintering. The presence of the supernal Alphaproteobacteria, Bifidobacteriales, and Lactobacillaceae in the gut of honeybees fed sucrose and cheaper than honey both indicate that sucrose is very suitable as the overwintering food for honeybees.