Numerous studies have suggested that nuclear factor-κB (NF-κB) and inducible nitric oxide synthase (iNOS) are important mediators of inflammatory response in human and animal models of arthritis. Besides, oxidative stress markers, nitric oxide (NO) and peroxide (PO) are also major contributors in the pathogenesis of rheumatoid arthritis (RA). Over expression of these inflammatory mediators leads to the extracellular matrix degradation, and excessive cartilage and bone resorption, ultimately leading to the irreversible damage to joints. The aim of the present study was to investigate the anti-arthritic mechanism of bioflavonoids, rutin and rutin-conjugated gold nanoparticles (R-AuNPs) by determining their role in the modulation of NF-κB and iNOS expression in collagen-induced arthritis (CIA) model of rats. Arthritis was induced by the subcutaneous administration of bovine type II collagen. Treatment was started with rutin, indomethacin+rutin (I+R) and R-AuNPs on the day of CIA induction. The severity of arthritis was determined by measuring the arthritic score on alternate days until mean arthritic score of 4 was observed. The NO and PO levels were also analyzed in serum samples. NF-κB and iNOS expression levels were determined in spleen tissue samples by real time RT-PCR and immunohistochemistry. Marked reduction in the arthritic score as well as in the NO and PO levels was observed in the treated groups. A significant downregulation in the NF-κB and iNOS expression levels was also observed in the treatment groups compared to the arthritic control group. Collectively, the findings suggest potential clinical role of rutin and R-AuNPs in the treatment of rheumatoid arthritis.