In many mammals, after semen deposition, a subpopulation of the sperm is transported to the lower oviduct, or isthmus, to form a functional sperm reservoir that provides sperm to fertilize oocytes. The precise molecular interactions that allow formation of this reservoir are unclear. It is proposed that binding of sperm receptors (lectins) to their oviductal cell ligands is accomplished by glycans. Previous results indicated that Lewis trisaccharides are present in glycosphingolipids and O- and N-linked glycans of the porcine isthmus and that Le^X-containing molecules bind porcine sperm. Immunohistochemistry indicated that the Lewis structures identified by mass spectrometry were, in fact, Lewis X (Le^X) trisaccharides. These motifs were localized to the luminal border of the isthmus. Assays using fluoresceinated glycans showed that 3-O-sulfated Le^X (suLe^X) bound to receptors localized on the head of nearly 60% of uncapacitated boar sperm but that the positional isomer 3-O-sulfo-Le^A (suLe^A) bound to <5% of sperm. Sperm also bound preferentially to suLe^X made insoluble by coupling to beads. Capacitation reduced the ability of suLe^X to bind sperm to <10%, perhaps helping to explain why sperm are released at capacitation. Pretreatment of oviduct cell aggregates with the Le^X antibody blocked 57% of sperm binding to isthmic aggregates. Blocking putative receptors on sperm with soluble Le^X and suLe^X glycans specifically reduced sperm binding to oviduct cells up to 61%. These results demonstrate that the oviduct isthmus contains Le^X-related moieties and that sperm binding to these oviduct glycans is necessary and sufficient for forming the sperm reservoir.