A circannual periodicity in thyrotropin (TSH) secretion has been reported but the causes of these phenomenon are still undefined. Vitamin D exerts a direct influence on pituitary axes including the hypothalamus-pituitary-thyroid axis. Aims of the present study were to investigate the presence of a seasonal variability of TSH secretion and to study the association between vitamin D status and TSH levels in a population of euthyroid adults. For this purpose, we recruited 294 euthyroid adults (M/F 133/161, 48.5 ± 12.4 years). Study participants underwent clinical examination and routine biochemistry assessment. Vitamin D deficiency was diagnosed for serum 25(OH) vitamin D <25 nmol/l. Significantly higher TSH levels were found in subjects who underwent blood sampling during the Autumn-Winter compared with individuals evaluated in Spring-Summer (2.3 ± 1.3 vs. 1.8 ± 1.1 μIU/ml, p = 0.03). Vitamin D deficiency was strongly associated with higher TSH levels (p = 0.01) after adjusting for sex, age, and sample's season. Although vitamin D deficiency was also associated with metabolic syndrome and its components, the association between TSH levels and vitamin D status persisted also considering these confounders. These data reveal the occurrence of seasonal variability of serum TSH concentration in euthyroid subjects and provide evidence for the first time that an association exists between vitamin D status and serum TSH levels.