<p>The oviduct is an important reproductive organ that fosters fertilization and early embryo development in mammals. With growing utilization of assisted reproductive technologies (ARTs) in animal agriculture, recent advancements in cell culture modeling have the potential to optimize in vitro fertilization methods. Organoids are a three-dimensional (3D) cell culture model poised to serve a key role in our understanding of endogenous oviductal physiology in cattle. However, limited information is currently available for the use of bovine oviductal organoids. Thus, the objective of our study was to conduct a comprehensive characterization of bovine oviductal organoids including polarity, cryopreservation, responsiveness to hormonal cues, and extracellular vesicle assessment. We observed that bovine oviductal organoids have the capacity for culture longevity for at least 86&#xa0;days with or without cryopreservation. Furthermore, the organoid polarity can be manipulated by presence or absence of an extracellular matrix, and they respond to stimulation with hormones associated with estrus and diestrus through gene expression changes and extracellular vesicle secretion. In conclusion, bovine organoids display similar characteristics to in vivo oviductal cells and constitute a cell culture model in which to study oviductal physiology to improve ARTs in beef and dairy agriculture.</p>