The γ-aminobutyric acid type A receptor (GABAAR) is negatively modulated by two structurally similar neurosteroids, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS). This study attempted to ascertain the molecular mechanisms of inhibition of the GABA-ergic current by neurosteroids. We demonstrated that the presence of the γ subunit in GABAAR enhances the efficacy of DHEAS without altering its binding affinity. A saturating concentration of DHEAS blocked approximately 75 % of currents mediated by GABAAR, which is composed of human α1, β1, and γ2S subunits, whereas the inhibition was only 35 % in GABAAR containing only α1 and β1 subunits. The IC50 values of DHEAS with and without the γ subunit were almost identical. In contrast to DHEAS, neither the affinity nor the efficacy of PS was altered by the γ subunit. When Val256 of α1 subunit was mutated to Ser, the mutant channel became resistant to inhibition by both DHEAS and PS. PS exerted its inhibitory effect by enhancing the desensitization kinetics of GABAAR possibly through promoting the interaction between the M2-M3 linker and extracellular loop 7/loop 2. Mutant α1, containing double Cys in loop 2/loop 7 and the M2-M3 linker, formed disulfide bonds three times as much fast, when treated with saturating GABA+PS, compared with GABA alone or with GABA+DHEAS. We demonstrated that PS, but not DHEAS, mediates GABA-ergic inhibition by promoting collisions between the structural elements involved in receptor desensitization, i.e., loop 2, loop 7, and the M2-M3 linker, thus following different inhibitory mechanisms. © 2015, Springer Science+Business Media New York.