Progesterone (P₄) induces a membrane depolarization and various ion fluxes (chloride efflux, sodium and calcium influxes), which are required for the human sperm acrosome reaction (AR). By use of the potentiometric fluorescent dye DiSC₃(5) and two different technical approaches, the present study aimed to quantify and further analyze P₄-induced modifications in membrane potential in capacitated human spermatozoa. Spectrofluorimetric analysis revealed that the mean resting membrane potential of sperm was −58 ± 2 mV (n = 12). When 10 μM P₄ was added, the sperm membrane depolarized by ∼ 15 mV, partly driven by a Cl⁻ efflux. It subsequently repolarized to reach a significant lower potential than the initial resting potential in two thirds of the tested samples. The flow cytometry analysis showed a heterogeneous resting membrane potential and revealed that the depolarization-hyperpolarization events concerned only subpopulations, between 3% and 40% of the sperm cells according to the samples (n = 7). We hypothesize that P₄ has a beneficial effect on the ability of zona pellucida to promote the AR in a sperm subpopulation by increasing the number of hyperpolarized cells presenting a membrane potential that is compatible with the opening of T-type calcium channels by subsequent zona pellucida-induced depolarization.