The aim of the present study was to establish new reference intervals for serum thyrotropin (TSH) levels in Japanese subjects without antithyroid antibodies. We reviewed the serum TSH level of all patients 20 years of age and over who attended the outpatient clinic of our hospital between January 1, 2003, and September 20, 2010. The thyroid gland of every patient was examined by ultrasonography, and subjects found to have a normal thyroid were chosen. The following subjects were excluded: subjects with past history of thyroid diseases; subjects whose serum was positive for antithyroid antibodies; pregnant women; patients taking medication that might affect their free thyroxine (fT₄) level or TSH levels. Ultimately, 1388 subjects were included in the reference population. The serum TSH levels shifted to higher ranges as the age of the groups increased. The calculated reference range was 0.39-4.29 mIU/L in the 20-29-year-old group, 0.34-3.90 mIU/L in the 30-39-year-old group, 0.56-5.02 mIU/L in the 40-49-year-old group, 0.51-5.30 mIU/L in the 50-59-year-old group, 0.60-4.85 mIU/L in the 60-69-year-old group, 0.62-6.15 mIU/L in the over 70-year-old group. The results of this study showed that the upper limit of the normal range of serum TSH levels increased with age in a Japanese population. Since the number of elderly reference subjects was relatively small, further study is needed. Setting the age- and race-specific reference limits for serum TSH levels is important in order to prevent significant misclassifications of patients with abnormal TSH levels.