Abstract: Nesfatin-1 is the hypothalamus gene encoding nuclear protein-2(nucleobindin-2, NUCB2) produces a neuropeptide after cracking. Studies have shown that nesfatin-1 play an important role in reproductive regulation, in order to study the nesfatin-1 relation with Epinephelus coioides reproductive regulation, in this paper, we have cloned two NUCB2(NUCB2a and NUCB2b) gene, analyzes NUCB2 coding protein get sequence of amino acids about two nesfatin-1(nesfatin-1a and nesfatin-1b), at the same time, we analysis of the two kinds of nesfatin-1's signal peptide and mature peptide, found it is consistent with that of vertebrates. Through real-time quantitative PCR technology, we determined the mRNA expression level of Epinephelus coioides nesfatin-1 in different tissues. The expression patterns revealed that, nesfatin-1a and nesfatin-1b widely expressed in the brain tissue, especially nesfatin-1b abundantly expressed in the pituitary, also has the corresponding expression in the gonads. We determined thenesfatin-1 mRNA expression level of hypothalamus during ovary development, the result reveal that, nesfatin-1a and nesfatin-1b in the ovaries Ⅵ period(degradation of yolk formation stage oocytes) significantly increased, showed that two kinds of nesfatin-1 are likely to participate in the regulation of ovarian mature. The intraperitoneal injection experiment results show that, injecting nesfatin-1a and nesfatin-1b 15 min, both GnRH1 and GnRH3 mRNA expression level of the hypothalamus are reduce, but in the pituitary LH-βand FSH-βmRNA expression level has not changed. Injection of nesfatin-1a and nesfatin-1b 60 min, the mRNA expression level of the hypothalamus GnRH1 and GnRH3 still suppressed. Injected nesfatin-1a and nesfatin-1b after 60 min, there have no effect change on the expression level of LH-β, but significantly reduced the expression of FSH-β. Through the above study shows nesfatin-1a and nesfatin-1b involved in Epinephelus coioides reproductive regulation.