Sperm ion channels are associated with the quality and type of flagellar movement, and their differential regulation is crucial for sperm function during specific phases. The principal potassium ion channel is responsible for the majority of K+ion flux, resulting in membrane hyperpolarization, and is essential for sperm capacitation-related signaling pathways. The molecular identity of the principal K+channel varies greatly between different species, and there is a lack of information about boar K+channels. We aimed to determine the channel identity of boar sperm contributing to the primary K+current using pharmacological dissection. A series of Slo1 and Slo3 channel modulators were used for treatment. Sperm motility and related kinematic parameters were monitored using a computer-assisted sperm analysis system under non-capacitated conditions. Time-lapse flow cytometry with fluorochromes was used to measure changes in different intracellular ionic concentrations, and conventional flow cytometry was used to determine the acrosome reaction. Membrane depolarization, reduction in acrosome reaction, and motility parameters were observed upon the inhibition of the Slo3 channel, suggesting that theSlo3gene encodes the main K+channel in boar spermatozoa. The Slo3 channel was localized on the sperm flagellum, and the inhibition of Slo3 did not reduce sperm viability. These results may aid potential animal-model-based extrapolations and help to ameliorate motility and related parameters, leading to improved assisted reproductive methods in industrial livestock production.