Methylmercury (MeHg) is a ubiquitous environmental pollutant, which can cross the placenta and blood brain barrier, thus affecting fetal growth and development. Although previous studies have demonstrated that MeHg induces endoplasmic reticulum (ER) stress in rat cerebral cortex and primary neurons, the role of ER stress in MeHg-induced neurodevelopmental toxicity remains unclear. Here, we used ICR pregnant mice and hippocampal neurons cells (HT22 cells) to investigate the molecular mechanism by which MeHg exposure during pregnancy affects neurodevelopment. We found that prenatal MeHg exposure caused developmental delay in offspring, accompanied with ER stress, cell apoptosis, cell cycle arrest and abnormal DNA methylation. Then, we used ER stress specific inhibitor 4-PBA and CHOP siRNA to investigate the role of ER stress on HT22 cells damage caused by MeHg. The results showed that 4-PBA pretreatment restored MeHg-induced axonal shortening and alleviated apoptosis, cell cycle arrest and DNA methylation. At the same time, the activation of CHOP/c-Jun/GADD45A signaling pathway was inhibited, and the interaction between CHOP and c-Jun was weakened. In addition, CHOP siRNA reduced the expression of c-Jun and GADD45A, and relieved DNA methylation levels to some extent. In summary, our study suggested that ER stress induced by MeHg mediated cell apoptosis and cell cycle arrest, and may affect DNA methylation through activation of CHOP/c-Jun/GADD45A signaling pathway, thus leading to neuronal damage.