Abstract The purpose of the present study was to identify a potential interference of bovine herpesvirus-1 (BoHV-1) with sperm–oocyte interactions during bovine in vitro fertilization. An inhibition of almost 70% of sperm–zona binding was observed when bovine cumulus-denuded oocytes were inseminated in the presence of 107 50% tissue culture infective dose/ml BoHV-1. The inhibitory effect of BoHV-1 on sperm–zona binding was mediated by an interaction of the virus with spermatozoa, but not with oocytes. Treatment of spermatozoa with BoHV-1, however, did not affect sperm motility and acrosomal status. Antiserum against BoHV-1 prevented the virus-induced inhibition of sperm–zona binding, indicating that BoHV-1 itself affects the fertilization process. In order to investigate which BoHV-1 glycoprotein(s) are responsible for the virus–sperm interaction, BoHV-1 was treated with monoclonal antibodies against the viral glycoproteins gB, gC, gD and gH prior to insemination. Anti-gC completely prevented the inhibitory effect of BoHV-1 on sperm–zona binding, while anti-gD caused a reduction of this inhibition. Further evidence for the involvement of gC and gD in the virus–sperm interaction was provided by the fact that purified gC and gD decreased sperm–zona binding in a dose-dependent way with gC being more effective than gD. These results indicated that BoHV-1 inhibits bovine sperm–zona binding by interacting with spermatozoa. The binding of BoHV-1 to a spermatozoon is mediated by the viral glycoproteins gC and gD, and therefore seems to be comparable with the mechanisms of BoHV-1 attachment to its natural host cell.