Among different physiological parameters, mammalian capacitation involves a plasma membrane hyperpolarization, which is necessary to acquire fertilization capability. However, this hyperpolarization event has not been completely demonstrated in human sperm. Briefly, capacitation is a complex physiological process which involves changes in plasma membrane composition, an increase in basal Ca²⁺ concentration, an intracellular alkalinization, phosphorylation of multiple proteins and the hyperpolarization of sperm membrane potential. In this report we show that a small percentage of human sperm indeed undergoes a plasma membrane hyperpolarization after in vitro capacitation, using flow cytometry. This hyperpolarization correlated with two other well-characterized capacitation parameters, such as increases in intracellular pH and Ca²⁺ concentrations. Sperm membrane hyperpolarization was completely inhibited by the addition of a high external K⁺ concentration (60 mM), indicating the participation of K⁺ channels. In order to identify the potential K⁺ channels which could be involved in this hyperpolarization, we performed a pharmacological characterization of this biological process using different K⁺ channel inhibitors including toxins which target Slo1 (charybdotoxin, slotoxin and iberiotoxin) or Slo3 channels (clofilium). Surprisingly our pharmacological results suggest that both members of the Slo family may potentially participate. In addition, we show that Slo3 channels are present in the human sperm membrane by immunoblotting. Consistently with the observed pharmacological profile, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 mM). Considered altogether, our data indicate that Slo1 and Slo3 could share a preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the hyperpolarization event.