The most extensively characterized human-derived cell line used in transcorneal permeability studies, in terms of passive transcellular and paracellular transport, transporter expression and metabolic enzymes, is the immortalized human corneal epithelial cell line (HCE-T). The purpose of this study is to describe the changes in the HCE-T barrier phenotype in vitro when valid cultivation conditions, in accordance with the standardized HCE-T cell-based model protocol, were employed. Evaluation of the structural and functional barrier properties revealed two different HCE-T barrier phenotypes, depending on the polycarbonate membrane pore size. Model I (pore size 0.4 μm) was characterized by a multilayered HCE-T epithelium at the apical side and a weak barrier function (70–115 Ω × cm2), whereas Model II (pore size 3 μm) consisted of an apical lipophilic HCE-T monolayer and a basolateral lipophilic monolayer of migrated HCE-T cells that showed improved barrier properties (1700–2600 Ω × cm2) compared with Model I. Considering the permeation of ophthalmic compounds and in vitro/ex vivo correlation, Model II was better able to predict transcorneal drug permeation. This study highlights the important aspects of HCE-T barrier phenotype variability that should be continuously monitored in the routine application of HCE-T cell-based models across both academic and pharmaceutical industry research laboratories.