Purpose: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease influenced by environmental triggers, including the commensal microbiota. Recent research has highlighted distinctive features of the gut microbiota in RA patients. This study investigates the therapeutic potential of berberine (BBR), a gut microbiota modulator known for its significant anti-RA effects, and elucidates the underlying mechanisms. Methods: Utilizing the collagen-induced arthritis (CIA) rat model, we comprehensively evaluated the anti-rheumatoid arthritis effects of BBR in vivo through various indices, such as paw edema, arthritis index, ankle diameter, inflammatory cytokine levels, pathological conditions, and micro-CT analysis. Employing network pharmacology, we identified potential targets involved in RA alleviation by BBR. To analyze comprehensive metabolic profiles and identify underlying metabolic pathways, we conducted a serum-based widely targeted metabolomics analysis utilizing LC-MS technology. An integrated network encompassing metabolomics and network pharmacology data was constructed using Cytoscape. The potential therapeutic targets and signaling pathways of BBR in the management of RA were predicted using network pharmacology. Key targets and pathways were further validated by molecular docking and immunofluorescent staining, which integrated findings from serum metabolomics and network pharmacology analysis. Additionally, we analyzed the gut microbiota composition in rats employing 16S rDNA sequencing and investigated the effects of BBR on the microbiota of CIA rats through bioinformatics and statistical methods. Results: Our results showed that BBR demonstrated significant efficacy in alleviating RA symptoms in CIA rats, as evidenced by improvements in paw redness and swelling, attenuation of bone and cartilage damage, reduction in synovial hyperplasia, inflammatory cell infiltration, and suppression of proinflammatory cytokines IL-1β, IL-6, IL-17A, and TNF-α. KEGG analysis highlighted the PI3K/AKT signaling pathway as a key mediator of BBR’s anti-RA effects. Metabolomics profiling via LC-MS revealed 22 potential biomarkers. Arginine and proline metabolism, cutin, suberine and wax biosynthesis, glycine, serine and threonine metabolism and taurine and hypotaurine metabolism are the most related pathways of BBR anti-RA. Molecular docking studies corroborated high affinities between BBR and key targets. Furthermore, 16S analysis demonstrated BBR’s capacity to modulate gut bacteria composition, including an increase in the abundance of Lachnoclostridium, Akkermansia, Blautia, Romboutsia, and Faecalibacterium genera, alongside a decrease in Prevotella_9 abundance in genus level. Integrated analysis underscored a strong correlation between serum microbiota and fecal metabolites. Conclusion: Our findings elucidate the multifaceted mechanisms underlying BBR’s therapeutic efficacy in RA, involving inhibition of the PI3K/AKT pathway, modulation of intestinal flora, and regulation of host metabolites. These insights provide novel perspectives on BBR’s role in RA management.