Human skin equivalents (HSEs) can be considered a valuable tool to study aspects of human skin, including the skin barrier, or to perform chemical or toxicological screenings. HSEs are three-dimensional skin models that are usually established using primary keratinocytes and closely mimic human skin. The use of primary keratinocytes has several drawbacks, including a limited in vitro life span and large donor-donor variation. This makes them less favorable for in vitro toxicity screenings. Usage of an established keratinocyte cell line circumvents these drawbacks and enables the generation of easy-to-generate and reproducible HSEs, which can be used for pharmacological and/or toxicological screenings. For such screenings, a proper barrier function is required. In this study, we investigated the barrier properties of HSEs established with the keratinocyte cell line N/TERT (N-HSEs). N-HSEs showed comparable tissue morphology and expression of several epidermal proteins compared with HSEs established with primary keratinocytes. Our results clearly demonstrate that N-HSEs not only contain several stratum corneum (SC) barrier properties similar to HSEs, including the presence of the long periodicity phase and a comparable SC permeability, but also show some differences in lipid composition. Nonetheless, the similarities in barrier properties makes N/TERT cells a promising alternative for primary keratinocytes to generate HSEs.