In our previous study, we explored the therapeutic effect of berberine (BBR) against IL-21/IL-21R mediated inflammatory proliferation of adjuvant-induced arthritic fibroblast-like synoviocytes (AA-FLS) through the PI3K/Akt pathway. The current study was designed to explore the therapeutic potential of BBR (15–45 µM) against IL-21/IL-21R mediated autophagy in AA-FLS mediated through PI3K/Akt signaling and Th17/Treg imbalance. Upon IL-21 stimulation, AA-FLS expressed elevated levels of autophagy-related 5 (Atg5), Beclin-1 and LC3-phosphatidylethanolamine conjugate 3-II (LC3-II) through the utilization of p62 and inhibition of C/EBP homologous protein (CHOP). BBR (15–45 µM) inhibited autophagy in AA-FLS cells mediated through PI3K/Akt signaling via suppressing autophagic elements, p62 sequestration and induction of CHOP in a dose-dependent manner. Moreover, IL-21 promoted the uncontrolled proliferation of AA-FLS through induction of B cell lymphoma-2 (Bcl-2) and diminished expression of Bcl-2 associated X protein (BAX) via PI3K/Akt signaling. BBR inhibited the proliferation of AA-FLS via promoting apoptosis through increased expression of BAX and diminished Bcl-2 transcription factor levels. Furthermore, T cells stimulated with IL-21 induced CD4+ CD196+ Th17 cells proliferation through RORγt activation mediated in a PI3K/Akt dependent manner. BBR inhibited the proliferation of Th17 cells through downregulation of RORγt in a concentration-dependent manner. BBR also promoted the differentiation of CD4+ CD25+ Treg cells through induction of forkhead box P3 (Foxp3) activation via aryl hydrocarbon receptor (AhR) and upregulation of cytochrome P450 family 1, subfamily A, polypeptide 1 (CYP1A1). Collectively, we conclude that BBR might attenuate AA-FLS proliferation through inhibition of IL-21/IL-21R dependent autophagy and regulates the Th17/Treg imbalance in RA. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.