Epithelial tight junction (TJ) is a barrier that separates the body from the external environment and regulates paracellular trafficking of macromolecules. TJ is composed of a multi-protein complex including several transmembrane proteins such as claudins, occludin, and junctional adhesion molecules. Among them claudin is a key protein to regulate the paracellular permeability. MA026 is an N-terminal acylated cyclic depsipeptide with 14 amino acid residues, which was isolated based on an anti-hepatitis C virus activity in 2013 from pseudomonas sp. RtIB026 living in gastrointestinal tract of rainbow trout. Recently, it is also reported that MA026 has an irreversible inhibitory activity to the TJ opening via the binding to claudin-1 [1]. In the development of new non-invasive TJ opener that overcomes the challenges in administering biopharmaceuticals through epithelium while suppressing the entry of foreign compounds, we focused on MA026 and planned to perform its structure-activity relationship (SAR) study. However, our synthesized MA026 failed to concur with its native structure and did not show any TJ opening activity. Through the synthesis of a large number of MA026 derivatives and their HPLC retention time (RT) analysis, then NMR and MS analyses, we found a new depsipeptide 1 with the same physicochemical property as the natural product. Depsipeptide 1 showed the TJ opening activity with a similar potency to the natural one. The N-terminal modification and alanine scanning of 1 revealed the important structural factors for the activity. Based on these findings, further SAR study for understanding the mode of action of MA026 to claudins and developing the potent non-invasive TJ opener is currently underway.