<p>Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that promotes the exchange of chloride (Cl⁻) and bicarbonate (HCO₃⁻), modulating the balance of ions and water in various tissues. CFTR is also expressed in the equatorial region of the spermatozoa’s head. However, the modulation of ionic and water transport via CFTR in spermatozoa needs further investigation. To better understand how CFTR participates in the molecular mechanisms behind spermatozoa modulation of water transport, and its impact on sperm physiology, CFTR function of human spermatozoa was inhibited with 20 µM CFTR_Inh-172. The inhibition of CFTR promoted a decrease in intracellular [Cl⁻] and membrane glycerol/water permeability, without any impact on sperm vitality or off-target effect in Ca²⁺ channels (assessed through the evaluation of intracellular [Ca²⁺]). To reverse the effects of CFTR inhibition, we incubated spermatozoa with CFTR-carrying seminal fluid extracellular vesicles (SF-EVs). We reported that the SF-EVs were able to restore the CFTR activity of spermatozoa previously exposed to the inhibitor, characterized by a restoration of Cl⁻, glycerol/water permeability, and a recovery of sperm osmoregulation capacity. Our results provide evidence for the important role of CFTR function in sperm osmoregulation and suggest that the use of CFTR-containing EVs - already employed in the treatment of cystic fibrosis - could be explored to enhance sperm quality.</p>