Gonadotropin-releasing hormone (Gnrh) plays important roles in reproduction by stimulating luteinizing hormone release, and subsequently ovulation and sperm release, ultimately controlling reproduction in many species. Here we report on a new role for this decapeptide. Surprisingly, Gnrh3-null zebrafish generated by CRISPR/Cas9 exhibited a male-biased sex ratio. After the dome stage, the number of primordial germ cells (PGCs) in gnrh3(-/-) fish was lower than that in wildtype, an effect that was partially rescued by gnrh3 overexpression. A terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) analysis revealed no detectable apoptosis of PGCs in gnrh3(-/-) embryos. Proliferating PGCs could be detected in wild-type embryos, while there was no detectable signal in gnrh3(-/-) embryos. Compared with wild type, the phosphorylation of AKT was not significantly different in gnrh3(-/-) embryos, but the phosphorylation of ERK1/2 decreased significantly. Treatment with a Gnrh analog (Alarelin) induced ERK1/2 phosphorylation and increased PGC numbers in both wild-type and gnrh3(-/-) embryos, and this was blocked by the MEK inhibitor PD0325901. The relative expression of sox9a, amh, and cyp11b were significantly upregulated, while cyp19a1a was significantly downregulated at 18 days post-fertilization in gnrh3(-/-) zebrafish. Taken together, these results indicate that Gnrh3 plays an important role in early sex differentiation by regulating the proliferation of PGCs through a MAPK-dependent path.