Accurate spatial and temporal expression of gonadotrope-specific genes, such as the gonadotropin-releasing hormone receptor (GnRHR) gene, is critical for gonadotrope maturation. Herein, we show that a specific E-box in the mouse GnRHR promoter binds two group A basic-helix-loop-helix (bHLH) transcription factors. Mutation of this E-box decreases expression in mouse gonadotrope-derived alphaT3-1 and LbetaT2 cell lines. Microarray and western blots show that the bHLH transcription factor NeuroD1 is strongly expressed in the gonadotrope progenitor, alphaT3-1, whereas Mash1 is strongly expressed in the more mature gonadotrope, LbetaT2. Over-expression of NeuroD1 or Mash1 increases expression of the GnRHR gene or a multimer of the E-box and this increase is lost upon mutation of the E-box. Electrophoretic mobility shift assays reveal that the GnRHR E-box binds NeuroD1 from alphaT3-1 cells, but binds Mash1 from LbetaT2 cells. The sequential binding of different members of the group A bHLH transcription factor family to mouse GnRHR E-box 3 as the gonadotrope differentiates may represent a mechanism necessary for proper spatial and temporal expression of the GnRHR during gonadotrope development.