Gonadotropin‐releasing hormone (GnRH) I and II are hypothalamic decapeptides with pivotal roles in the development of reproductive competence and regulation of reproductive events. In this study, transcriptional regulation of the human GnRH II gene was investigated. By scanning mutation analysis coupled with transient promoter assays, the motif at −641/−636 (CATGCC, designated GII‐Sil) was identified as a repressor element. Mutation of this motif led to full restoration of promoter activity in TE671 medulloblastoma and JEG‐3 placenta choriocarcinoma cells. Supershift and chromatin immunoprecipitation assays showed in vitro and in vivo binding of NF‐κB subunit p65 and the retinoic acid receptors, RARα and RXRα, to the promoter sequences. Over‐expression of these protein factors indicated that p65 is a potent repressor, and the RARα/RXRα heterodimer is involved in the differential regulation of the GnRH II gene in neuronal and placental cells. This was confirmed by quantitative real‐time PCR. Treatment of cells with the RARα/RXRα ligands, all‐trans retinoic acid and 9‐cis‐retinoic acid, reduced and increased GnRH II gene expression in TE671 and JEG‐3 cells, respectively. Taken together, these data demonstrate the differential roles of NF‐κB p65 and RARα/RXRα, interacting with the same sequence in the promoter of the human GnRH II gene to influence gene expression in a cell‐specific manner.