Mammalian spermatozoa are unable to fertilize an oocyte immediately after their deposit in the female genital tract. To gain competence to fertilize, they must undergo a gradual maturation process termed capacitation that is accompanied by: 1) sperm intracellular alkalinization, evoked by proton extrusion through proton channels; 2) elevation of the intracellular [Ca2+], evoked by calcium influx through calcium ion channels, and 3) membrane hyperpolarization that is evoked by K+ efflux. Whereas the proton and calcium channels of human sperm are identified as Hv1 and CatSper, the identity of the principal potassium channel of human sperm (hKSper) was unknown. Potassium channels are indispensable for normal sperm physiology as they regulate cell membrane potential and cell motility. Recently, a pH-sensitive sperm K+ channel, encoded by the Slo3 gene was shown to be regulated by intracellular alkalinization and essential for male fertility in mice. It has been assumed, but never proven, that the K+ channel of human sperm has a similar molecular identity. Here we present full electrophysiological and pharmacological characterization of hKSper in both mature and developing human spermatozoa, and report that the molecular identity of hKSper is different from that of mouse KSper. In human sperm, the KSper current is conveyed by the big conductance, calcium-activated potassium channel (Slo1) which differs from mouse Slo3 in both its regulation and functional relationship with other sperm ion channels.