Variations in ambient temperature () may significantly influence the gut microbiotas of ectothermic and endothermic animals, affecting fitness. It remains unclear, however, whether temperature fluctuations affect the gut microbial communities of hibernating animals during torpor. To investigate temperature-induced changes in the gut microbiota during hibernation under entirely natural conditions, we took advantage of two adjacent but distinct populations of the least horseshoe bat (Rhinolophus pusillus), which inhabit sites with a similar summer but a different winter . Using 16S rRNA gene high-throughput sequencing, we estimated differences in gut microbial diversity and composition between the hibernating (winter) and active (summer) populations at both sites. During the active period, gut microbiotas did not differ significantly between the two populations, probably due to the similar s. However, during hibernation, a higher was associated with decreased α-diversity in the gut microbiome. During hibernation, temperature variation did not significantly affect the relative abundance of , the dominant phylum at both sites, but marked site-specific differences were detected in the relative abundances of , , and . In total, 74 amplicon sequence variants (ASVs) were significantly differentially abundant between the hibernating and active bat guts across the two sites; most of these ASVs were associated with the cooler site, and many belonged to pathogenic genera, suggesting that lower ambient temperatures during hibernation may increase the risk of pathogen proliferation in the host gut. Our findings help to clarify the mechanisms underlying the gut microbiota-driven adaptation of hibernating mammals to temperature changes. Temperature variations affect gut microbiome diversity and structure in both ectothermic and endothermic animals. Here, we aimed to characterize temperature-induced changes in the gut microbiotas of adjacent natural populations of the least horseshoe bat (Rhinolophus pusillus) which hibernate at different ambient temperatures. We found that the ambient temperature significantly affected the α-diversity, but not the β-diversity, of the gut microbiota. Bats hibernating at cooler temperatures experienced more drastic shifts in gut microbiome structure, with consequent effects on energy-related metabolic pathways. Our results provide novel insights into the effects of ambient temperature on the gut microbiotas of hibernating animals.