9th International Workshop on the Biology of Fish Gametes, 15-18 July 2024, León, Spain, Spermatozoon volume regulation is an essential determinant of male fertility competence in mammals and oviparous fishes. In mammals, aquaporin water channels (AQP3 and -7 and -8) have been suggested to play a role in spermatozoon cell volume regulatory responses. In contrast to mammals, the ejaculated spermatozoa of marine teleosts experience a high hypertonic shock in seawater, resulting in an Aqp1aa-mediated water efflux, cell shrinkage and the activation of motility. However, the potential involvement of other aquaporins in cell volume regulatory mechanisms in post-activated marine spermatozoa are unknown. Using the gilthead seabream (Sparus aurata) as a model of marine teleosts and a paralog-specific antibody, we show that the AQP3 ortholog Aqp3a is highly accumulated in the mid posterior region of the spermatozoon flagella. Aqp3a-mediated water conductance is specifically inhibited by a recently developed AQP3 antagonist (DFP00173) as well as by the Aqp3aspecific seabream antibody (¿-Aqp3a) when expressed in Xenopus laevis oocytes. Both DFP00173 and ¿-Aqp3a impair post-activated sperm motility kinetics (% motility, % progressivity, and curvilinear velocity) with respect to the controls at 30 s post activation in a dose-dependent manner. Interestingly, in a close resemblance to the phenotypes of AQP3-deficient murine sperm, field emission scanning and transmission electron microscopy revealed that both Aqp3a inhibitors induce an abnormal sperm tail morphology, including local swelling and tail angulation, with complete coiling of the flagella in some cases. These data suggest that Aqp3a may function as an osmosensor to regulate cell volume under a high hypertonic stress, or that the channel is required for the efflux of specific solutes which can inhibit sperm motility, Supported by the Spanish Ministry of Science, Innovation and Universities (PID2022-138066OB-I00 to J.C., BES-2017-080778 to J.C-A., and CEX2019-000928-S to ICM), Peer reviewed