Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide involved in the regulation of reproductive function in vertebrates. It is able to inhibit the synthesis and secretion of GnRH and Gth in the HPG axis. However, the regulatory role and mechanism by which current gonadal steroid hormones regulate GnIH are still unclear. In this study, transcription factor binding site analysis was performed on the promoter sequence of zebrafish GnIH. Whereafter, transgenic zebrafish (GnIH: mCherry) accurately labeled GnIH and esr2b knockout zebrafish, which were constructed previously, were used to explore the regulation between estrogen and GnIH. In vitro exposure of wild-type zebrafish embryos and transgenic zebrafish embryos to estradiol showed that 10 μM estradiol significantly increased the transcription level of GnIH. However, both 10 μM and 50 μM estradiol significantly increased the transcription level of GnIH in esr2b knockout zebrafish. We compared the expression levels of GnIH in esr2b knockout zebrafish and wild-type zebrafish at different developmental stages (48 hpf–120 hpf). The results showed that from 96 hpf, the expression level of GnIH in esr2b knockout zebrafish was significantly higher than that in wild-type zebrafish, indicating that esr2b is involved in the negative regulation of GnIH, and this regulatory relationship is established on the fourth day of zebrafish development.