In addition to its role in adaptive thermogenesis, brown adipose tissue (BAT) may protect from weight gain, insulin resistance/diabetes, and metabolic syndrome. Prior studies have shown contradictory results regarding the influence of thyroid hormone (TH) levels on BAT volume and activity. The aim of this pilot study was to gain further insights regarding the effect of TH treatment on BAT function in adult humans by evaluating the BAT mass and activity prospectively in six patients, first in the hypothyroid and then in the thyrotoxic phase. The study subjects underwent 18F-fluorodeoxyglucose positron emission tomography/computed tomography (PET/CT) scanning after cold exposure to measure BAT mass and activity while undergoing treatment for differentiated thyroid cancer, first while hypothyroid following TH withdrawal at the time of the radioactive iodine treatment and then three to six months after starting TH suppressive treatment when they were iatrogenically thyrotoxic. Thermogenic and metabolic parameters were measured in both phases. All study subjects had detectable BAT under cold stimulation in both the hypothyroid and thyrotoxic state. The majority but not all (4/6) subjects showed an increase in detectable BAT volume and activity under cold stimulation between the hypothyroid and thyrotoxic phase (total BAT volume: 72.0 ± 21.0 vs. 87.7 ± 16.5 mL, p = 0.25; total BAT activity 158.1 ± 72.8 vs. 189.0 ± 55.5 SUV*g/mL, p = 0.34). Importantly, circulating triiodothyronine was a stronger predictor of energy expenditure changes compared with cold-induced BAT activity. Iatrogenic hypothyroidism lasting two to four weeks does not prevent cold-induced BAT activation, while the use of TH to induce thyrotoxicosis does not consistently increase cold-induced BAT activity. It remains to be determined which physiological factors besides TH play a role in regulating BAT function.