Veterinarians use flumequine (FLU) widely but its toxicological effects are still unclear. FLU doses of 53, 200, or 750 mg/kg were administered orally for six weeks to pubertal male rats for evaluation of their toxicity. Weight gain was poorer after seven days of exposure to FLU 750, but relative weights of the brain, adrenal and thyroid glands, and testes were notably higher. Haematological and lipid profile parameters, cardiac markers, and inorganic phosphate significantly increased in the FLU 750 group. Blood glucose, oestradiol and serum concentrations of immunoglobulins G (IgG) and E (IgE) significantly decreased after treatment. The levels of interleukins 10 (IL-10) and 6 (IL-6) fell significantly in the FLU 200 and FLU 750 groups. Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) and cyclooxygenase-2 (Cox-2) expression amplified after treatment. Serum levels of free triiodothyronine (fT3) and free thyroxine (fT4) reduced in the FLU 200 and FLU 750 groups without changes in total T3 or T4 level. All doses of FLU significantly depressed concentrations of thyroid-stimulating hormone (TSH) and testosterone. Histopathology of thyroid glands from rats treated with FLU 750 showed degeneration and depletion of thyroid follicular epithelial cells. Expression of 8-hydroxydeoxyguanosine (8-OHdG) was increased in a dose-dependent manner in the brain, but decreased in the testes. Expression of CYP1A1 increased in the adrenal and pituitary glands. The results of this study suggest that the toxicity of FLU in rats is an effect of its disruptive influence on the pituitary-thyroid hormonal system and on the dysfunction of the immune system.