Background <p>Chloride ions (Cl) regulate sperm physiology, influencing spermatogenesis, volume regulation, capacitation, and fertilization processes. They contribute to the maintenance of membrane potential and intracellular pH, both of which are critical for sperm motility and capacitation. Any deviation in Cl homeostasis causes impaired sperm function and male infertility. Although several Cl channels and transporters have been implicated in the Cl homeostasis and osmoregulation of sperm cells, the precise mechanisms and molecular components governing volume regulation during sperm development remain unclear.</p> Methods <p>We used a combination of electrophysiological recordings via the patch-clamp technique and biochemical analyses, including western blotting and immunocytochemistry, to demonstrate the functional expression of a novel chloride channel, Chloride Intracellular Channel 4 (CLIC4), in the plasma membrane of sperm cells. To assess physiological roles, we analyzed sperm cells from wild type and null mutant mice (<i>clic4</i>^{<i>−/−</i>}), measuring motility, morphology and acrosome reaction.</p> Results <p>We identified previously uncharacterized IAA-94-sensitive chloride currents in mouse sperm cells. Genetic ablation of CLIC4 eliminated these IAA-94-sensitive currents. Notably, CLIC4 regulates cell volume during sperm maturation without altering membrane potential, motility, or the acrosome reaction. CLIC4 activity in sperm cells is modulated by Protein Kinase C (PKC).</p> Conclusion <p>CLIC4 is a key component of the sperm cell volume regulation machinery, modulating Cl fluxes during maturation. These findings provide new insights into the molecular basis of sperm osmoregulation and may inform therapeutic strategies for male infertility.</p>