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Simple Summary: Père David's deer (Elaphurus davidianus) are classified as extinct in the wild from the International Union for Conservation of Nature Red List. In 2021, there were more than 6000 individuals in Dafeng Reserve, China. With the increasing of population size for Père David's deer in Dafeng Reserve, it is urgently needed to conduct disease research in order to protect this animal better. Diarrhea is one of the most common diseases affecting the health of Père David's deer. However, little is known about how the intestinal ecology changes in these diarrheic animals. The aim of this study is to reveal the changes of intestinal microbiome and metabolic pathways in diarrheic Père David's deer based on gut microbiome and metabolic pathways. Using 16S rRNA gene sequencing and ultra-high performance liquid chromatography combined with tandem mass spectrometry, the gut microbiota and fecal metabolites were analyzed in five diarrheic Père David's deer. Results demonstrated the distinct changes in the diversity and composition of gut microbiota, as well as great changes in numerous fecal metabolic profiles in diarrheic Père David's deer. The integrated pathway analysis revealed serious disturbances in several metabolic pathways, such as lipid, bile acid, cofactor and vitamin metabolism. These data provided important gut ecology information for diarrheic Père David's deer, which may facilitate improved diagnostic and treatment strategies for sick animals in the future. Diarrhea is one of the most common diseases affecting the health of Père David's deer (Elaphurus davidianus). It is believed that an imbalanced intestinal ecology contributes to the etiology of the condition. However, little is known about how the intestinal ecology changes in these diarrheic animals. In this study, 16S rRNA gene sequencing and ultra-high performance liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) were used to investigate the gut microbiota and fecal metabolites in five Père David's deer with diarrhea. The results showed that when compared with healthy individuals, considerable changes in the gut microbiome were observed in diarrheic animals, including a significant reduction in microbial diversity and gut microbiota composition alterations. Furthermore, the profiles of numerous fecal metabolites were altered in diarrheic individuals, showing large-scale metabolite dysregulation. Among metabolites, acylcarnitines, lysophosphatidylcholine, bile acids, and oxidized lipids were elevated significantly. Constantly, several metabolic pathways were significantly altered. Interestingly, predicted metabolic pathways based on 16S rRNA gene sequence and differential metabolite analysis showed that lipid metabolism, cofactor, and vitamin metabolism were altered in sick animals, indicating microbiota-host crosstalk in these deer. When combined, the results provide the first comprehensive description of an intestinal microbiome and metabolic imbalance in diarrheic Père David's deer, which advances our understanding and potential future treatment of diarrheic animals. [ABSTRACT FROM AUTHOR]