It is unclear whether hypothyroidism is present in patients with Prader-Willi syndrome (PWS). This study aimed to clarify the state of the hypothalamic-pituitary-thyroid axis and the effects of growth hormone (GH) treatment on thyroid function in pediatric patients with PWS. We retrospectively evaluated thyroid function in 51 patients with PWS before GH treatment using a thyroid-releasing hormone (TRH) stimulation test (29 males and 22 females; median age, 22 months). We also evaluated the effect of GH therapy on thyroid function by comparing serum free triiodothyronine (fT3), free thyroxine (fT4), and thyroid stimulating hormone (TSH) levels at baseline, 1 year, and 2 years after GH therapy. TSH, fT4, and fT3 levels were 2.28 μU/ml (interquartile range [IQR]; 1.19-3.61), 1.18 ng/dl (IQR; 1.02-1.24), and 4.02 pg/dl (IQR; 3.54-4.40) at baseline, respectively. In 49 of 51 patients, the TSH response to TRH administration showed a physiologically normal pattern; in two patients (4.0%), the pattern suggested hypothalamic hypothyroidism (delayed and prolonged TSH peak after TRH administration). TSH, fT4, and fT3 levels did not change significantly during 1 or 2 years after GH treatment. The TSH response to TRH showed a normal pattern in most patients, and thyroid function did not change significantly during the 2 years after initiating GH treatment.