Dental fluorosis (DF) is one of the important performances of endemic fluorosis. Some studies indicated that estrogen receptor (ESR) gene polymorphisms were associated with bone metabolism-related diseases. Therefore, it is possible that the variation in ESR genotypes will be associated with DF status. A case-control study was conducted among children aged 8-12 years with (n = 75) or without (n = 165) DF in China to investigate the relationship between ESR gene polymorphisms and DF. Gene polymorphisms were genotyped using the PCR-RFLP procedure. Children carrying R allele of ER RsaI had significantly increased risk of DF (Odds ratio (OR) = 1.821; 95% confidence interval (CI), 1.013-3.274) compared to children carrying r allele of ER RsaI in endemic fluorosis villages. For children with high-loaded fluoride status, carrying X allele of ESRα XbaI had a significantly decreased risk of DF (OR = 0.542; 95% CI, 0.314-0.936) compared to carrying x allele. This study provides the first evidence of an association between polymorphisms in the ESR gene with DF in high-fluoride-exposed populations. Further studies are needed to confirm the association.