NR0B1 gene is an atypical orphan nuclear receptor which expresses specifically in the hypothalamus of both humans and mice. NR0B1 has conserved functions in regulating gonadal development and sexual determination in vertebrates. However, little is known about the functions of NR0B1 gene in ventral hypothalamus. In this study, we use zebrafish that is readily amenable to genetic manipulations as the model system to study nr0b1gene functions. We screened out nr0b1 knockout line t313 and the transgene line s313. s313 specifically labels the nr0b1 neurons in the ventral hypothalamus. Microarray and fluorescence in situ hybridization (FISH) show pdyn gene expression in nr0b1 neurons of ventral hypothalamus. Knockdown or knockout nr0b1 gene leads to up-expression of pdyn gene, whereas overexpression of nr0b1 gene leads to decreasing expression of pdyn gene in the ventral hypothalamus of 2dpf larva. Knockout nr0b1 in 8dpf larva and 3 month adult zebrafish also indicate nr0b1 can inhibit pdyn and oprk1expression. These results suggest that zebrafish nr0b1 can repress the expression of pdyn gene in ventral hypothalamus.