Prebiotics are widely used to modulate gut microbial communities, but their effects on the honey bee gut microbiota remain poorly understood. Xylo-oligosaccharides (XOS) are recognized as potential prebiotic substrates in humans and livestock, whereas their effects in honey bees have rarely been investigated in vivo. In this study, we evaluated the effects of dietary XOS supplementation on the gut microbiota of Apis mellifera. Total bacterial load and the absolute and relative abundances of five core bacterial groups (Lactobacillus Firm-5, Bombilactobacillus, Bifidobacterium, Gilliamella, and Snodgrassella alvi) were quantified using spike-in-based absolute 16S rRNA gene sequencing. Microbial diversity and community structure were further assessed using alpha-diversity indices and beta-diversity analyses. XOS supplementation did not significantly affect total bacterial abundance or the abundance of the five core bacterial groups (p > 0.05). No significant differences were detected in alpha diversity (p > 0.05), and the control and XOS groups exhibited largely overlapping community structures based on unweighted and weighted UniFrac analyses. However, PICRUSt2 analysis identified differences in several predicted microbial functional pathways between the two groups (p < 0.05). These results indicate that dietary XOS supplementation had limited detectable effects on the abundance, diversity, and structure of the established honey bee gut microbiota under the tested conditions, although changes in its predicted functional potential were observed. Our findings suggest that the efficacy of prebiotics in honey bees may depend on host-specific microbial ecology, and that prebiotic responses observed in mammalian systems may not be directly applicable to honey bees. These results highlight the importance of validating commercial prebiotic products using bee-specific in vivo evidence before their widespread application.