Our previous study demonstrated the involvement of cystic fibrosis transmembrane conductance regulator (CFTR) in transporting bicarbonate that is necessary for sperm capacitation; however, whether its involvement is direct or indirect remains unclear. The present study investigated the possibility of a Cl⁻/HCO₃^– exchanger (solute carrier family 26, number 3 [SLC26A3]) operating with CFTR during guinea pig sperm capacitation. Incubating sperm in media with various concentrations of Cl⁻ resulted in varied percentages of capacitated sperm in a concentration-dependent manner. Depletion of Cl⁻, even in the presence of HCO₃⁻, abolished sperm capacitation and vice versa, indicating the involvement of both anions in the process. Capacitation-associated HCO₃⁻-dependent events, including increased intracellular pH, cAMP production, and protein tyrosine phosphorylation, also depend on Cl⁻ concentrations. Similar Cl⁻ dependence and inhibitor sensitivity were observed for sperm-hyperactivated motility and for sperm-egg fusion. The expression and localization of CFTR and SLC26A3 were demonstrated using immunostaining and Western blot analysis. Taken together, our results indicate that Cl⁻ is required for the entry of HCO₃⁻ that is necessary for sperm capacitation, implicating the involvement of SLC26A3 in transporting HCO₃⁻, with CFTR providing the recycling pathway for Cl⁻.