Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by persistent inflammation, cartilage degradation, and bone erosion, mediated primarily by pro-inflammatory cytokines such as interleukin-6 (IL-6) and matrix metalloproteinase-9 (MMP-9). This study aimed to investigate the anti-inflammatory and chondroprotective potential of Indian mackerel (Rastrelliger kanagurta) fish oil, a rich source of omega-3 polyunsaturated fatty acids (PUFAs), in a collagen-induced arthritis (CIA) rat model. Twenty adult female Wistar rats were divided into four groups: normal control, CIA control, CIA + indomethacin (0.5 mg/day), and CIA + fish oil (1 mL/day orally). The extracted fish oil, analyzed by GC–MS, contained high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Administration of fish oil significantly reduced arthritis scores, paw thickness, and joint swelling, accompanied by improvements in joint histopathology compared with the untreated CIA group. ELISA results showed a marked decrease in serum IL-6 and MMP-9 levels (p < 0.05), indicating attenuation of systemic inflammation and joint matrix degradation. These findings demonstrate that R. kanagurta fish oil possesses potent anti-inflammatory and tissue-protective effects, suggesting its potential as a natural, sustainable therapeutic adjunct for managing rheumatoid arthritis.