The gut microbiota of bats is vital for their roles in health and the ecosystem, yet studies on hibernating bats in southwest China, particularly in the unique karst landscape of Guizhou, are limited. We captured three hibernating bat species-Pipistrellus (PB), Rhinolophus (RB), and Myotis (MB)-in Liping County, collecting rectal samples for 16S rRNA amplicon sequencing. Data processing involved Trimmomatic, Flash, and Qiime2 for operational taxonomic unit (OTU) standardization and species annotation via the Greengenes database. Differential abundance was analyzed using LEfSe, and diversity metrics were assessed through alpha and beta diversity analyses. The RB group was predominantly composed of (80.99%), while MB and PB exhibited diverse compositions with significant OTU richness (729 in MB). Notable genera included and in RB and in MB. High proportions of unclassified taxa were observed, particularly in RB (83.81%). Functional predictions indicated metabolic pathways, with a significant representation of human diseases in PB. Culturomics revealed the successful cultivation of and from bats for the first time and appears to have identified a new bacterium that is likely closely related to .IMPORTANCEOur research reveals significant differences in the composition and diversity of the gut microbiota among three bat groups (PB, MB, and RB) from Guizhou. While Proteobacteria predominates in all groups, its abundance varies. Notably, the high richness of operational taxonomic units (OTUs) in the MB group suggests a more diverse microbial ecosystem, underscoring the complex interactions between species diversity, diet, gut microbiota, and overall ecological dynamics in bats. Furthermore, the substantial presence of unknown bacterial species in their intestines highlights the critical importance of cultivation-based approaches. The presence of specific taxa may have potential health implications for both bats and humans. These findings emphasize the need for further investigations into the functional roles of these microbiota and their contributions to host health. Future research should focus on longitudinal studies to elucidate these intricate interactions.