Endocrine-disrupting chemicals (EDCs), such as perfluorooctanoic acid (PFOA), have been linked to adverse effects on reproductive health. However, the impact of PFOA exposure during the embryonic stage on hypothalamic gonadotropin-releasing hormone (GnRH) neurons, which are central to the regulation of the hypothalamic–pituitary axis, that controls the reproductive system, has not been investigated. In this study, immortalized embryonic mouse hypothalamic cells (mHypoE-N46) were used to evaluate alterations in GnRH expression following PFOA exposure in vitro. In addition, the expression levels of Pnx, Gpr173, BDNF, and Ntrk2—upstream signals known to regulate GnRH expression—were examined. mHypoE-N46 cells were treated with PFOA for 2-24 hours, and gene expression and protein expression levels were evaluated. PFOA significantly altered the gene expression levels of Pnx, Gpr173, BDNF and Ntrk2. GnRH expression was significantly increased at both the mRNA and protein levels. In conclusion, exposure to PFOA may perturb the upstream signaling of GnRH and may directly or indirectly affect the expression of GnRH, suggesting that PFOA may alter the GnRH regulatory network.