The mechanism by which Bovine herpesvirus 1 (BHV-1) induces apoptic cell death is not fully understood. Attachment but not penetration of BHV-1 is necessary to induce apoptosis in target cells suggesting that one or more BHV-1 envelope glycoprotein(s) could be involved in the activation of the apoptotic process. In this context, we show that BHV-1 virions devoid of glycoprotein D (gD) are no longer able to induce apoptosis. In contrast, virions which contain gD in the viral envelope but do not genetically encode gD (BHV-1 gD-/+) induce comparable level of apoptosis as wild type (wt) BHV-1. In addition, monoclonal antibodies directed against gD strongly reduced the high levels of apoptosis induced by wt BHV-1 and BHV-1 gD-/+ but not the background level of apoptosis induced by BHV-1 gD-/-. This demonstrate that the induction of apoptosis is directly due to BHV-1 viral particles harboring gD in the viral envelope. Altogether, these results provide evidence for the involvement of gD in the mechanism by which BHV-1 induces apoptosis. This could have important implications in the development of new, more effective and safer vaccines and also help to better understand the pathogenesis of BHV-1.