Rheumatoid arthritis (RA) is a multifactorial autoimmune disease that affects the joints of approximately 1 % of the population worldwide and is seen 2–4 times less in males. INSL3 is a gender-specific peptide hormone produced much higher in males than in females and may have an anti-inflammatory role in RA. So, in this study, it was aimed to determine the possible anti-inflammatory effect and dose of insulin-like factor-3(INSL3) in an experimental Complete Freund’s adjuvant(CFA)-induced RA male rat model and lipopolysaccharide(LPS)-induced macrophage cell line and compare it with prednisolone therapy. For in vivo experiments, 48 male mice were randomly divided into 6 groups with 8 subjects in each group: Control group, Arthritis group, Arthritis + Prednisolone(10 mg/kg) group, Arthritis + INSL3(0.08–0.8–8 μg/day) groups. Joint tissue samples obtained from sacrificed subjects were examined by histochemical and immunohistochemically methods after biometric analyses, arthritis severity scoring, and thermal latency experiments. LPS-induced macrophage cells were also treated with prednisolone(1 µg/ml) and INSL3(50–100-200 nM). Cell viability, cell morphology, and TNF-α and IL-6 immune reactivity were evaluated. According to the data obtained from in vivo analyses, it was seen that INSL3 reduced the paw diameter and arthritis severity scoring, degenerative changes, and inflammation and increased the thermal latency, compared to the arthritis group, although not as much as the prednisolone treatment group. In vitro analyses showed that high doses of INSL3 had positive effects on cell viability, morphology, TNF-α, and IL-6 immune reactivity. In conclusion, it was determined that the anti-inflammatory effect of INSL3 was not as pronounced as prednisolone, but it had a more favorable impact on macrophage cell viability and morphology. It was concluded that INSL3 may be a protective therapeutic agent in combination with existing treatment protocols in treating many autoimmune diseases.