MicroRNAs have recently gained popularity because of their critical roles in the regulation of many biological processes. Their expressions are found to be attributed to different inflammatory disorders such as rheumatoid arthritis (RA). RA is a chronic autoimmune disorder that could be triggered due to genetic and epigenetic factors. Berberine (BBR) is an isoquinoline derivative alkaloid with a long history of medicinal use. It has wide anti-inflammatory, antibacterial, and antioxidative activities. However, the effect of BBR on miRNA-146a-5p (miR-146a-5p) expression level has not yet been studied. Thus, the present study is the first report that explores the exact mechanism of BBR on modulating miR-146a-5p expression in adjuvant induced arthritis (AIA) model. The arthritis severity scoring was assessed using hematoxylin-eosin and toluidine blue staining. Furthermore, immunohistochemical staining for the CD68+ macrophages expression was performed. MiR-146a-5p expression level was measured by reverse transcriptase quantitative real time polymerase chain reaction (qRT-PCR). The obtained results showed downregulation of miR-146a-5p associated with increased levels of antinuclear autoantibodies (ANA), tumor necrosis factor-α (TNF-α), IL-1β, nuclear factor kappa B (NF-κB), IL-6, interleukin-1 receptor-associated kinase 1 (IRAK1), TNF receptor-associated factor 6 (TRAF6) and quantitative expression of CD68+ macrophages in AIA rat model. Remarkably, BBR treatment restores the levels of all these mediators. Collectively, the current results revealed the possible role of miR-146a-5p in the regulation of RA disease by manipulating TRAF6/IRAK1/NF-kB signaling as a macrophage inflammatory pathway. Furthermore, BBR showed potential ability to restore expression levels of miR-146a-5p and modulate NF-κB pathway proteins in AIA rat model.