As with other mammalian species, human spermatozoa experience a decrease in extracellular osmolarity in cervical mucus upon ejaculation, which requires the efflux of osmolytes and water to counteract swelling that hinders mucus penetration. Recent evidence for the operation of K⁺ channels in the process of volume regulation suggests parallel involvement of Cl⁻/anion channels for electro-neutrality as in somatic cells. This was studied using ejaculated spermatozoa washed at seminal osmolality and incubated for 30 min in a medium of mucus osmolality in the presence of Cl⁻ channel blockers. Increases in cell size measured as laser forward-scatter by flow cytometry were detected in the presence of 100 μM 5-nitro-2(3-phenylpropylamino) benzoic acid, 400 μM diisothiocyanato-stilbene-2,2′-disulphonic acid, and 20 μM tamoxifen. No volume changes were found with 400 μM 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulphonic acid, 200 μM verapamil, or niflumic acid, whereas 1 mM niflumic acid induced shrinkage. Among the candidate channel proteins, Western blotting revealed the presence of ClC-3 (CLCN3) at 87 kDa, but the absence of ClC-2 (CLCN2) from sperm proteins in all samples tested. ICln (CLNS1A) was found in only one of eight samples. Immunocytochemistry localized CLCN3 to the sperm tail. To confirm molecular identities, sperm mRNA was extracted and checked for quality by the presence of protamine 2 transcripts and the absence of sperm DNA and leukocyte mRNA using reverse transcription-polymerase chain reaction. Transcripts of Clcn3 were found in all samples and that of Clns1a in some but not all samples. Clcn3 was therefore considered the most likely candidate of Cl⁻ channel involved in volume regulation of human sperm.