Neutrophil extravasation is central to inflammatory skin diseases like psoriasis and atopic dermatitis. In vivo, neutrophils have been shown to migrate through cell-to-cell junctions (paracellular pathway) or directly through the body of the endothelial cell (transcellular pathway). In vitro, however, neutrophil migration is a largely paracellular process where cells preferentially cross at tricellular corners devoid of tight junctions. To approximate the type of cells encountered by extravasating neutrophils in vivo, we developed a neutrophil-migration assay using primary human dermal microvascular endothelial cells. We show here that a large proportion of migrating neutrophils traverse a monolayer of microvascular endothelium using a purely transcellular pathway. In addition, we demonstrate that F-actin is rearranged similarly in neutrophils undergoing diapedesis along either route. This in vitro model closely simulates the physiological process of neutrophil extravasation in vivo and can be further utilized to evaluate the relative contribution of distinct migratory pathways to the pathophysiology of inflammatory skin disease.