The current study investigated the therapeutic effect of berberine (BBR), an alkaloid derivative against IL-21/IL-21R mediated phosphotidyl inositol 3 kinase/protein kinase B (PI3K/Akt) signaling in adjuvant induced arthritic fibroblast-like synoviocytes (AA-FLS) isolated from rats and IL-21 mediated osteoclastogenesis in bone-marrow derived monocytes (BMMs). BBR (15–45 μM) treatment attenuated the gene and protein levels of IL-21R complex. BBR suppressed the levels of IL-21 (20 ng/ml) mediated production of inflammatory cytokines such as: tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and interleukin 23 (IL-23) in AA-FLS cells. Subsequently, BBR ameliorated the gene and protein expression levels of mechanistic target of rapamycin (mTOR), IL-23 and nuclear factor kappa B (NFκB) p65 through the inhibition of PI3K and upregulation of phosphatase and tensin homolog (PTEN) at the protein level. Furthermore, BBR also inhibited the phosphorylation of Akt and NFκB p65 in a dose dependant manner. LY294002 (20 μM) treatment suppressed the PI3K/Akt signaling and its downstream elements in AA-FLS cells. BBR also modulated IL-21 mediated osteoclastogenesis through the suppression of PI3K dependant nuclear factor of activated T-cells 1 (Nfatc1) induction. Moreover, BBR controlled the osteoclast differentiation via inhibition of various bone resorptive enzymes including: cathepsin K, matrix metalloproteinase 9 (MMP9) and tartarate acid phosphatase (TRAP). LY294002 also inhibited osteoclast formation via suppression of PI3K mediated Nfatc1 induction and other downstream elements. Overall, our findings suggest that BBR is a potential candidate for therapeutic targeting of IL-21/IL-21R mediated RA pathogenesis.