Bisphenol A (BPA) is a widespread endocrine disrupting chemical strongly suspected to have adverse health effects. Numerous tissues and cells are affected by BPA, and we showed recently that BPA targets include ameloblasts and enamel. We therefore investigated the effects of BPA on ameloblasts and the possible involvement of the estrogen signaling pathway. Rats were exposed daily to low-dose BPA from the conception to 30 days after birth (30 D). Seventy-five percent of 30 D rats exposed to BPA developed enamel hypomineralization similar to human Molar Incisor Hypomineralization (MIH). Ameloblast proliferation was induced by BPA in vivo and in vitro and could lead to enamel hypomineralization by affecting amelogenesis process. The proliferation of the rat dental epithelial cell line HAT-7 was also increased by estrogen (E2). Ameloblasts express ERα but not ERβ both in vivo and in vitro. The ER antagonist ICI 182 780 was used to inactivate ERα and abolished the effects of E2 on cell proliferation and transcription, but only partially reduced the effects of BPA. In conclusion, we show, for the first time, that: i) BPA has ER-dependent and ER-independent effects on ameloblast proliferation and gene transcription and ii) the estrogen signaling pathway is involved in tooth development and the enamel mineralization process. These results are consistent with the steroid hormones having effect on ameloblasts, raising the issues of the hormonal influence on amelogenesis and possible differences in enamel quality between sexes.