ABSTRACTBalantidium coli (B. coli) is a prevalent intestinal parasite in monkeys, significantly impacting their health. Previous studies have demonstrated that B. coli infection in pigs leads to severe dysregulation of the gut microbiota. However, there has been no report on the alterations in fecal microbiota and metabolites in rhesus monkeys infected with B. coli. In order to investigate the differences in gut microbiota and metabolites between healthy rhesus monkeys and those infected with B. coli, we conducted gene sequencing and gas chromatography–mass spectrometry (GC–MS) analysis of fecal samples from 6 healthy rhesus monkeys and 5 rhesus monkeys infected with B. coli. The results revealed significant differences in the composition of gut microbiota between rhesus monkeys infected with B. coli and healthy ones (p < 0.01). The abundance of Campylobacterota was significantly increased (p < 0.01), while the abundance of Bacteroidota was significantly decreased (p < 0.05). Prevotella 9 was the dominant genus in both groups, showing a significant increase in the infected group (p < 0.05). At the species level, Brachyspira hampsonii was significantly increased in the infected group (p < 0.01), whereas Prevotella copri, which was the dominant species in both groups, showed a significant decrease in the infected group (p < 0.05). Metabolomics studies indicated a significant decrease in levels of metabolites such as dihydrolipoamide, 9(Z),11(E)‐Conjugated Linoleic Acid, and 8,9‐DiHETrE within fecal samples from rhesus monkeys infected with B. coli (p < 0.05). Correlation analysis of the microbiome and metabolome suggested a close relationship between differential microbiota and metabolites. In conclusion, this study suggests that the colonization of B. coli is associated with dysbiosis of the monkey gut microbiota. This study provides a new insight that using intestinal microbes instead of antibiotics to treat balantidiosis can also serve as a reference for further research on the relationship between gut microbiota and metabolomics in host infections by other protozoa.