Primate beta-defensin 126 regulates the ability of spermatozoa to bind to oviductal epithelial cells in vitro. Bovine beta-defensin 126 (BBDI26) exhibits preferential expression in the cauda epididymis of the bull, but there have been few studies on its functional role in cattle. The aim of the present study was to examine the role of BBD126 in bull sperm binding to bovine oviductal epithelial cell (BOEC) explants. BBD126 has been shown to be highly resistant to the standard methods of dissociation used in other species and, as a result, corpus epididymal spermatozoa, which have not been exposed to the protein, were used to study the functional role of BBD126. Corpus epididymal spermatozoa were incubated with recombinant (r) BBD126 in the absence or presence of anti-BBD126 antibody. Addition of rBBD126 significantly enhanced the ability of epididymal spermatozoa to bind to BOEC explants (P < 0.05). Anti-BBD126 antibody blocked the BBDI 26-mediated increase in sperm binding capacity. Ejaculated spermatozoa, which are coated with native BBD126 protein but also a large number of seminal plasma proteins in vivo, were incubated with rBBD126 in the absence or presence of the anti-BBD126 antibody. Addition of rBBDI26 significantly enhanced the ability of ejaculated spermatozoa to bind to BOEC explants (P < 0.05), whereas rBBD126 also reduced corpus sperm agglutination (P < 0.05). These results suggest that, similar to the role of its analogue in the macaque, spermatozoa with more BBD126 in their acrosome may represent spermatozoa with more oviduct binding capacity.