Abstract: GPR54 is the cognate receptor of Kisspeptin, which plays a significant role in fish reproduction regulation by acting on the Gonadotropin-Releasing Hormone (GnRH). In order to study the regulation of GnRH by Kisspeptin/GPR54 system in the Dabry’s sturgeon (Acipenser dabryanus), the full-length cDNAs of two gpr54 genes were cloned, which were designated as dsgpr54-1 and dsgpr54-2, encoding 379 and 368 amino acids, respectively. Multiple amino acid sequences alignment and evolutionary tree analysis indicated that Gpr54 of Dabry’s sturgeon shared higher sequence identities and closer evolutionary distances with its counterparts in tetrapods. By quantitative real-time PCR, it showed that dsgpr54-1 were transcribed in both gonad (testis and ovary) and brain (hypothalamus, pituitary, mesencephalon and telencephalon) with the highest transcription level in the hypothalamus. Conversely, dsgpr54-2 was only found in the brain, with high expression levels in the pituitary, mesencephalon and hypothalamus. In order to investigate whether gpr54 could regulate the expression of gnrh in the hypothalamus by combined to its ligand Kisspeptin, decapeptides of both Kiss-1 and Kiss-2 (10 nmol/L and 1000 nmol/L) of Dabry’s sturgeon were synthesized and injected to the peritoneal cavities of 9 month juveniles. It showed by quantitative real-time PCR that both the two doses of Kiss-1 and Kiss-2 injection induced the increase of gpr54 expression, and 10 nmol/L Kiss-2 injection increased the transcription of dsgpr54-2 significantly (P<0.05). Besides, both two doses of Kiss-1 injection reduced thegnrh expression. However, 10 nmol/L Kiss-2 injection caused the increase of the gnrh1 expression and the decrease of gnrh2 expression, while in the 1000 nmol/L Kiss-2 injection group, gnrh1 expression decreased with no changes of the gnrh2 transcription. The above results revealed that both the two gpr54 genes could bind to its ligands kiss-1 and kiss-2, but with receptor-ligand selection discrepancy. Kiss-1 and Kiss-2 regulate the gnrh expression differently by activating their receptor GPR54 in the hypothalamus of Dabry’s sturgeon.