A 2.5-year-old female English setter was presented with clinicopathological findings suggestive of hypothyroidism. Thyroid-stimulating hormone (TSH: 4.3 ng/ml, normal <0.5), but also free thyroxine (FT4: 77 pmol/l, normal 7.7–48), total thyroxine (67 nmol/l, normal 17–58) and total triiodothyronine (8.7 nmol/l, normal 0.3–3.2) measured with a chemiluminescent immunoassay (CLIA) were elevated. Further investigations revealed oval-shaped hypoechoic thyroid glands characteristic of thyroiditis and very high titres for thyroglobulin autoantibodies, T4 autoantibodies and T3 autoantibodies. As expected the FT4 concentration determined by equilibrium dialysis (ED) to exclude analyte-dependent interferences was low. With levothyroxine therapy clinical signs disappeared and the concentrations of TSH and FT4 (ED) were within the respective reference intervals again. This case demonstrates that anti-thyroid hormone antibodies can cause erroneous CLIA readings, and that their presence should be clarified if thyroid hormone results do not match the clinical presentation or are discordant with TSH results.