BACKGROUND: Rheumatoid arthritis-associated interstitial lung disease (RA-ILD) is considered one of the most severe complications of rheumatoid arthritis. The etiology of RA-ILD is complex, involving genetic and environmental factors. Recent studies suggest that the gut microbiota, a critical component of the immune system, may influence the pathogenesis of RA and other autoimmune disorders. However, specific data on the gut microbiota in patients with RA-ILD remain limited. OBJECTIVE: This study aimed to investigate alterations in the gut microbiota of RA-ILD patients and compare these profiles with those of RA patients without ILD and health controls. METHODS: We included three groups: RA-ILD patients (n = 30), RA patients without ILD (n = 31), and health controls (n = 30). Fresh fecal samples were collected and subjected to 16S rRNA gene sequencing to analyze microbial diversity. Statistical analyses involved α-diversity and β-diversity assessments, principal coordinates analysis (PCoA), and differential abundance testing with LEfSe and PICRUSt2. RESULTS: Significant differences in gut microbiota composition were observed between RA-ILD patients and the other groups. Notably, g_Prevotella showed differential abundance, particularly in RA-ILD patients. KEGG pathway analysis revealed upregulation in several metabolic pathways in RA-ILD compared to RA and health controls, suggesting a distinct microbial metabolic activity associated with RA-ILD. CONCLUSION: Our findings indicate that RA-ILD patients have a markedly different gut microbiota profile compared to RA patients without ILD and health controls. The observed microbial alterations may contribute to RA-ILD pathogenesis and could serve as potential biomarkers or therapeutic targets. Further studies are needed to explore these findings' clinical implications and validate the role of gut microbiota in RA-ILD progression.