The substantia nigra pars reticulata (SNR) is involved in movement and seizure control. In male but not female postnatal day 15 (PN15) rats, GABA_A receptor agonists depolarize the SNR neurons and increase the expression of the calcium‐regulated gene KCC2 (potassium/chloride cotransporter). Moreover, in PN15 rat SNR, 7β‐estradiol down‐regulates KCC2 expression only in the presence of depolarizing GABA_A receptor responses. The hypothesis tested here was that GABA_A receptors and estradiol also regulate the expression of the phosphorylated form of the transcription factor cAMP responsive element binding protein (phosphoCREB), in PN15 rat SNR and substantia nigra pars compacta (SNC). Rats were injected with muscimol or 17β‐estradiol or their vehicles, and killed 1 h later. Sections were stained with an antibody specific for phosphoCREB alone or counterstained with either tyrosine hydroxylase (TH)‐ or parvalbumin (PRV)‐specific antibodies. Muscimol increased phosphoCREB‐ir in male but not in female SN neurons. Using gramicidin perforated patch clamp of PN14–15 SNC neuron, it was shown that muscimol bath application depolarized male SNC neurons but did not significantly alter membrane potential in females. In males, 17β‐estradiol decreased phosphoCREB expression in all studied cell types. In females, 17β‐estradiol did not influence phosphoCREB expression in PRV‐ir SNR cells, but increased it in the dopaminergic SN neurons. These data suggest that GABA_A receptor activation and estradiol promote the sexual differentiation of the SN in a cell‐type‐specific manner, by influencing calcium‐regulated gene transcription, and therefore promoting the acquisition of sex‐specific roles of the SN in movement and seizure control.