Human spermatozoa encounter an osmotic decrease from 330 to 290 mOsm l?1 when passing through the female reproductive tract. We aimed to evaluate the role of chloride channels in volume regulation and sperm motility from patients with asthenozoospermia. Spermatozoa were purified using Percoll density gradients. Sperm volume was measured as the forward scatter signal using flow cytometry. Sperm motility was analyzed using computer?aided sperm analysis (CASA). When transferred from an isotonic solution (330 mOsm l?1) to a hypotonic solution (290 mOsm l?1), cell volume was not changed in spermatozoa from normozoospermic men; but increased in those from asthenozoospermic samples. The addition of the chloride channel blockers, 4,4′?diisothiocyanatostilbene?2,2′?isulfonic acid (DIDS) or 5?nitro?2?(3?phenylpropylamino) benzoic acid (NPPB) to the hypotonic solution caused the normal spermatozoa to swell but did not increase the volume of those from the asthenozoospermic semen. DIDS and NPPB decreased sperm motility in both sets of semen samples. The inhibitory effect of NPPB on normal sperm motility was much stronger than on spermatozoa from the asthenozoospermic samples. Both sperm types expressed ClC?3 chloride channels, but the expression levels in the asthenozoospermic samples were much lower, especially in the neck and mid?piece areas. Spermatozoa from men with asthenozoospermia demonstrated lower volume regulating capacity, mobility, and ClC?3 expression levels (especially in the neck) than did normal spermatozoa. Thus, chloride channels play important roles in the regulation of sperm volume and motility and are downregulated in cases of asthenozoospermia.