Rheumatoid arthritis (RA), an autoimmune disease, is characterized by chronic joint inflammation and pain. We previously found that the deletion of T-cell death-associated gene 8 (TDAG8) significantly reduces disease severity and pain in RA mice. Whether it is by modulating gut microbiota remains unclear. In this study, 64 intestinal samples of feces, cecal content, and cecal mucus from the complete Freund’s adjuvant-induced arthritis mouse models were compared. The α-and β-diversity indices of the microbiome were significantly lower in RA mice. Cecal mucus showed a higher ratio of Firmicutes to Bacteroidetes in RA than healthy mice, suggesting the ratio could serve as an RA indicator. Four core genera, Eubacterium_Ventriosum, Alloprevotella, Rikenella, and Treponema, were reduced in content in both feces and mucus RA samples, and could serve microbial markers representing RA progression. TDAG8 deficiency decreased the abundance of proinflammation-related Eubacterium_Xylanophilum, Clostridia, Ruminococcus, Paraprevotella, and Rikenellaceae, which reduced local mucosal inflammation to relieve RA disease severity and pain. The pharmacological block of the TDAG8 function by a salicylanilide derivative partly restored the RA microbiome to a healthy composition. These findings provide a further understanding of specific bacteria interactions with host gut mucus in the RA model. The modulation by TDAG8 on particular bacteria can facilitate microbiota-based therapy.