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The red panda (Ailurus fulgens) is a distinctive mammal known for its reliance on a diet primarily consisting of bamboo. The gut microbiota and overall health of animals are strongly influenced by diets and environments. Therefore, conducting research to explore the taxonomical and functional variances within the gut microbiota of red pandas exposed to various dietary and environmental conditions could shed light on the dynamic complexities of their microbial communities. In this study, normal fecal samples were obtained from red pandas residing in captive and semi-free environments under different dietary regimes and used for metabolomic, 16S rRNA, and metagenomic sequencing analysis, with the pandas classified into four distinct cohorts according to diet and environment. In addition, metagenomic sequencing was conducted on mucus fecal samples to elucidate potential etiological agents of disease. Results revealed an increased risk of gastrointestinal diseases in red pandas consuming bamboo shoots due to the heightened presence of pathogenic bacteria, although an increased presence of microbiota-derived tryptophan metabolites appeared to facilitate intestinal balance. The red pandas fed bamboo leaves also exhibited a decrease in gut microbial diversity, which may be attributed to the antibacterial flavonoids and lower protein levels in leaves. Notably, red pandas residing in semi-free environments demonstrated an enriched gut microbial diversity. Moreover, the occurrence of mucus secretion may be due to an increased presence of species associated with diarrhea and a reduced level of microbiota-derived tryptophan metabolites. In summary, our findings substantiate the influential role of diet and environment in modulating the gut microbiota of red pandas, offering potential implications for improved captive breeding practices., (© 2024 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)