We have identified a short peptide sequence (L-R5) acting as partial inhibitor of intracellular protein kinase C, capable of tight junction modulation in terms of reversible and non-toxic drug permeation enhancement. L-R5 is a pentapeptide with a cell-penetrating group at the N-terminus and of the sequence myristoyl-ARRWR. Apically applied in vitro, L-R5 transiently increased epithelial permeability within minutes, enhancing apical-to-basolateral (AB) transport of 4-kDa dextran and BCS class III drug naloxone. L-R5 was shown to be stable and effective at 37°C over a period of 24 hours. L-R5 was shown to be non-cytotoxic in consecutive exposure studies on primary human nasal epithelial cells by LDH release assay and ciliary beating frequency test. Finally, L-R5 by itself showed very low diffusion across epithelial monolayers, which is of advantage with regard to its expected negligible systemic bioavailability and side effects. Taken together, these data demonstrate the potential of short peptide partial inhibitor L-R5 to enhance the epithelial paracellular permeability via a reversible mechanism, and in a non-toxic manner.