Introduction <p>Regulatory B cells (Bregs) play a pivotal role in the immune modulation of rheumatoid arthritis (RA). The levels of CD19⁺CD24^hiCD38^hi Bregs in peripheral blood and their association with clinical indicators in RA patients have not been extensively studied. The aim of this study was to investigate the alterations in peripheral blood CD19⁺CD24^hiCD38^hi Breg levels in RA patients and their association with clinical indicators. Additionally, we explored the variations in Breg levels before and after treatment.</p> Methods <p>Overall, 90 patients with newly diagnosed RA and 32 healthy controls were prospectively enrolled. Symptoms and laboratory findings were collected. Flow cytometry and three-color fluorescence antibody labeling were employed to detect CD19⁺CD24^hiCD38^hi Breg levels in peripheral blood mononuclear cells (PBMCs). The proportion of CD19⁺CD24^hiCD38^hi Bregs among mature B lymphocytes was analyzed, and correlations with clinical indicators were examined. A follow-up study of 26 RA patients was conducted to compare changes in Breg levels before and after six months of treatment.</p> Results <p>The levels of CD19⁺CD24^hiCD38^hi Bregs in RA patients were 89.63% of those in healthy controls. No significant differences in Breg levels were observed among patients with different disease activities. There was a positive correlation between CD19⁺CD24^hiCD38^hi Breg levels and morning stiffness duration (<i>r</i> = 0.227, <i>p</i> = 0.027), and negative correlations with age and anti-CCP antibody titers (<i>r</i>=-0.234, <i>p</i> = 0.022; <i>r</i>=-0.218, <i>p</i> = 0.036). No significant correlations were found between Breg proportions and other clinical indicators. The follow-up study showed a decrease in disease activity scores and a significant increase in CD19⁺CD24^hiCD38^hi Breg levels after six months of treatment.</p> Conclusions <p>These findings suggest a potential association between Breg levels and the pathogenesis and inflammatory activity of RA. CD19⁺CD24^hiCD38^hi Bregs may serve as potential biomarkers for evaluating disease activity and treatment efficacy. Further research is warranted to explore the underlying biological mechanisms and clinical significance of Bregs in RA.</p>