Abstract:  MRL/MpJ‐Faslpr mice is widely accepted as a valuable model of systemic lupus erythematosus. As described in a previous work, the incidence of lupus in this strain is determined by sex hormones, i.e., estrogens and androgens. Moreover, we reported that the immunomodulatory action of melatonin in these mice was gender‐dependent probably through modulation and inhibition of sex hormones. Herein, we performed an experiment using hormone therapy, by treating female MRL‐lpr mice with testosterone and males with estradiol and with melatonin. A decrease in total serum immunoglobulin (Ig)G and IgM immunoglobulin titers, anti‐double‐stranded DNA, and anti‐CII autoantibodies in female mice treated with both melatonin and testosterone was revealed, along with an increase in pro‐inflammatory cytokines [interleukin (IL)‐2, IL‐6, interferon‐γ, tumor necrosis factor‐α, and IL‐1β), nitrite/nitrate and a decrease in anti‐inflammatory cytokines (IL‐10). Melatonin and estradiol treatment exhibited a similar effect in male mice. Autoantibody titer elevation and pro‐inflammatory versus anti‐inflammatory cytokine prevalence degraded all immunological parameters. Similar results were obtained when spleen and lymph node lymphocytes were cultured. Again, melatonin and testosterone treatment stimulated pro‐inflammatory and reduced anti‐inflammatory cytokines produced by lymphocytes in females. The effect was similar in males treated with melatonin and estradiol. In summary, we observed that although melatonin alone prevents lupus development in females, adding testosterone, increased pro‐inflammatory cytokine pattern. In contrary, estradiol‐treated males did not show any decrease in pro‐inflammatory cytokines but showed an increase in regard to melatonin controls. These findings confirm that melatonin action in MRL/MpJ‐Faslpr mice could be gender‐dependent through modulation of sex hormones.