Herpes simplex virus 1 (HSV-1) has to overcome skin or mucosa barriers to infect its human host. The impact of the various barrier functions on successful viral invasion is not known. Upon ex vivo infection of murine skin we observed efficient invasion only via the basal epidermal layer when the dermis was removed. Here, we investigated how wounding and intercellular junction formation control successful viral entry. After wounding of skin samples or removal of the stratum corneum, infected cells were rarely detected. Based on infection studies in epidermis from interferon-stimulated mice, we assume that mechanical wounding does not lead to an antiviral state that impedes infection. When we infected human skin equivalents we observed entry only into unstratified keratinocytes or after wounding of fully stratified cultures. Reduced infection of keratinocytes after calcium-induced stratification confirmed the impact of junction formation. To assess the effect of functional tight junctions, stratified cultures of Par3- or E-cadherin-deficient keratinocytes were infected. As the number of infected cells strongly increased with enhanced paracellular permeability, we conclude that the formation of functional tight junctions interferes with viral entry indicating that next to the stratum corneum tight junctions are a major physical barrier for HSV-1 invasion into tissue.