Levetiracetam (LEV), a novel anti-epileptic drug that has been demonstrated with an anti-inflammatory effect, but the exact mechanisms of its action remain to be fully defined. The present study aimed to evaluate the possible effects of LEV on lipopolysaccharides (LPS)-induced Janus kinase-signal transducers and activators of transcription (JAK/STAT) as well as toll-like receptor 4 (TLR4)/ mitogen activated protein kinase (MAPK) signaling pathways activation in adjuvant induced arthritis (AIA). Rats were allocated into normal control, three arthritic control groups: Complete Freund's Adjuvant (CFA) (0.4 ml/3days/12days), LPS (100 μg/kg/day), CFA+LPS, and three treated groups: CFA+LEV, LPS+LEV and CFA+LPS+LEV. LEV was administered in a dose 50 mg/kg/day for 15 day. After 28 days, tissue samples were collected for assessment of phosphorylated JAK2, STAT3, TLR4, MAPK and cathepsin K quantitative expression in synovium. Additionally, Serum samples were used for biochemical evaluation of interleukin-6 (IL-6), interleukin-1beta (IL-1B), LPS, anti-citrullinated protein antibody (ACPA) and 8-hydroxydeoxyguanosine (8-OHdG). Histopathological and macroscopical examinations of joints were also performed to support our study. Results indicated that LEV exerted its anti-inflammatory effect through inhibiting LPS-dependent phosphorylation of JAK2/STAT3 signaling. It significantly suppressed TLR4 and MAPK expressions, thereby decreasing release of inflammatory cytokines IL-1β, IL-6.LEV exhibited a potent inhibitory effect on cathepsin K and 8-OHdG parallel to confirmatory histopathological and macroscopical findings. In conclusion, LEV has a powerful therapeutic effect on adjuvant induced arthritis in rats and its mechanisms are strongly related to inhibiting excessive activation of JAK2-STAT3 and TLR4 pathways. This may add a new approach for treatment of RA.