The brain is a sexually dimorphic organ, with many sex differences arising during development due to the effect of gonadal hormones from embryonic day (E) 17. In this context, GABA exert trophic effects on immature neurons through GABAA receptors, whose functionality depends on its subunit composition and intracellular chloride levels. While many sex differences in GABA-mediated responses are attributed to gonadal hormones, few studies have examined potential differences prior to the critical period of brain masculinization. In this study, we investigated the functionality and expression of the GABAA receptor in sex-segregated hypothalamic neurons obtained from E15 mice and cultured for 3 days in vitro. Functional activity was assessed through calcium imaging by measuring the number of responsive neurons, amplitude, response kinetics, and decay following GABA exposure (10 μM). In parallel, qPCR analysis was performed to evaluate the mRNA expression of Nkcc1, Kcc2 (chloride co-transporters), and ε subunit receptor gene. We observed that female neurons reached their GABA response peak amplitude faster, with a greater proportion exhibiting fast decaying response compared to males. Gene expression analysis revealed no significant differences. These findings suggest that GABA responses are sexually dimorphic even in the absence of gonadal hormones, although this effect does not appear to be attributable to differences in the expression of Nkcc1, Kcc2 or GABAA receptor ε subunit.