The pulmonary route is gaining increasing popularity as a fast and effective way of delivering drugs to the lungs, both locally and systemically. Although the lung is naturally permeable to small-molecule drugs, the epithelial barrier and the presence of tight junctions (TJ) strictly regulate the absorption of large and polar molecules. It has been shown that polyunsaturated fatty acids (PUFAs) may integrate into membrane lipids and modify their physical properties by altering membrane fluidity and thus barrier function. In this study, the in vitro effect of PUFAs, specifically docosahexaenoic acid (DHA), linoleic acid (LA), arachidonic acid (AA), gamma- linoleic acid (GLA) and palmitoleic acid (PA), on airway epithelial cells (Calu-3 cells) with regards to their tight junction modulatory effect was investigated. Moreover, the effect of PUFAs on mucus production and inflammatory response, and the viscoelastic behaviour of airway mucus were also investigated. Calu-3 cells were treated with 100 μM of each PUFA and the transepithelial resistance (TEER) of Calu-3 cells, which is an in vitro measure of epithelial cell integrity, and paracellular epithelial permeability of Na fluorescein (paracellular marker) were measured. It was shown that the resistance dropped significantly after Calu-3 cells were incubated with PUFAs, compared to non-treated control cells. In general, the permeability increased following the rank order: GLA≥PA >DHA>AA>LA. Specifically, GLA and PA treatments enhanced paracellular transport of Na fluorescein 1.5 times over control. This increase in paracellular transport mirrored the concurrent drop in TEER values. PUFAs resulted in reduced mucus production in Calu-3 cells (except DHA) and did not result in cytotoxicity or inflammation (except AA) with the experiment concentration. It was concluded that PUFAs have the potential to modulate TJ and thus enhance the permeation of drugs through the lung epithelial cell barrier via the paracellular route.