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Tight junctions (TJs) play an important role in the maintenance of epithelial and endothelial barriers.Zonula occludens (ZO) proteins are scaffolding molecules essential for the formation of TJ complexes,and abnormalities in ZO proteins have been implicated in various T J-associated human diseases such as tumor invasion and metastasis,and barrier dysfunction.Recent studies reveal that liquid-liquid phase separation of ZO proteins drives the polymerization of TJ proteins into a continuous belt,which then recruits various proteins to form the TJ complex to regulate selective paracellular permeability and signal transduction.Herein,we describe recent advances on how ZO phase separation contributes to TJ formation and discuss the potential of phase separation as a target for the treatment of T J-associated diseases.