Sine oculis-related homeobox 3 (SIX3) and SIX6, 2 closely related homeodomain transcription factors, are involved in development of the mammalian neuroendocrine system and mutations of Six6 adversely affect fertility in mice. We show that both small interfering RNA knockdown in gonadotrope cell lines and knockout of Six6 in both embryonic and adult male mice (Six6 knockout) support roles for SIX3 and SIX6 in transcriptional regulation in gonadotrope gene expression and that SIX3 and SIX6 can functionally compensate for each other. Six3 and Six6 expression patterns in gonadotrope cell lines reflect the timing of the expression of pituitary markers they regulate. Six3 is expressed in an immature gonadotrope cell line and represses transcription of the early lineage-specific pituitary genes, GnRH receptor (GnRHR) and the common α-subunit (Cga), whereas Six6 is expressed in a mature gonadotrope cell line and represses the specific β-subunits of LH and FSH (LHb and FSHb) that are expressed later in development. We show that SIX6 repression requires interaction with transducin-like enhancer of split corepressor proteins and competition for DNA-binding sites with the transcriptional activator pituitary homeobox 1. Our studies also suggest that estradiol and circadian rhythm regulate pituitary expression of Six6 and Six3 in adult females but not in males. In summary, SIX3 and SIX6 play distinct but compensatory roles in regulating transcription of gonadotrope-specific genes as gonadotrope cells differentiate.