Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes functional disability due to bone destruction and severe joint pain. Current anti-rheumatic treatments develop severe complications and do not provide complete remission. Gold nanoparticles (AuNPs) have garnered attention because of their unique physical and chemical properties. In this study, we have evaluated the therapeutic effects of gold nanospheres (AuNSs) with two different ligands (targeted-nanoparticles) against collagen-induced arthritis (CIA) and compared the outcomes with conventional methotrexate (MTX) and biological (infliximab) treatments. Clinical evaluation was performed by radiographic and histological examinations. The bioaccumulation of AuNSs in vital organs was assessed. The mechanistic studies targeting pro-inflammatory/anti-inflammatory and angiogenic mediators' expressions were performed. Radiographic examination showed that the targeted AuNSs reduced joint space narrowing and bone erosion. Moreover, histopathological examination of rat ankle joints demonstrated that targeted AuNSs reduce bone and cartilage degeneration/inflammation. Gold nanospheres-conjugated with nucleus localized peptide (nuclear membrane-targeted) (AuNSs@NLS) has resolved bone destruction and inflammation compared to gold nanospheres-conjugated at polyethylene glycol (AuNSs@PEG). Although the AuNSs accumulated in different organs in both cases, they did not induce any toxicity or tissue damage. The two different targeted AuNSs significantly suppress inflammatory and angiogenic mediators' expression and induced anti-inflammatory cytokine production, but the AuNSs@NLS had superior therapeutic efficacy. In conclusion, these results suggested that nuclear membrane-targeted AuNSs effectively attenuated arthritis progression without systemic side effects.