Although estradiol (E2) plays a critical role in the promotion of pituitary development and in the regulation of various pituitary hormones, its effects on the thyroid-stimulating hormone (TSH) remain unaddressed. The actions of E2 are mediated by two classical nuclear estrogen receptors α (ERα) and β (ERβ) and the G protein-coupled estrogen receptor (GPER). However, the types of estrogen receptor involvement in the regulation of thyrotropes are still limited. In this study, we demonstrate that ERα, but not ERβ and GPER, is localized to thyrotropes in the pituitary of female mouse. In agreement with the presence of ERα in thyrotropes, E2 was shown to stimulate TSH release in vitro from primary culture of female mouse pituitary cells. PPT, a ERα-selective agonist, but not DPN (a ERβ-selective agonist) and G-1 (a GPER-selective agonist), was shown to stimulate TSH release in mouse pituitary cells. This effect could be prevented by the specific ER antagonist fulvestrant and the selective ERα antagonist MPP. The findings of this study suggest that E2 may bind to ERα to trigger TSH release and provide novel information on the differential regulation of multiple estrogen receptors in the pituitary.