Tight junctions (TJs) regulate paracellular permeability, cell polarity and cell mechanics of barrier-forming tissues. This Review explores how TJs adapt their structure and function across tissues, with a focus on the claudin-based strand network, its regulation by zonula occludens scaffold proteins and its dysfunction in human diseases. We discuss recent insights into TJ assembly through biomolecular condensation, highlighting how scaffold self-organization integrates adhesion, actin dynamics and polarity cues. We examine TJ maintenance across timescales, from rapid protein turnover to long-term remodelling during development. Finally, we focus on TJ function in lumen formation and review TJ dysfunctions and strategies to target TJ proteins therapeutically. We close by highlighting emerging approaches to tackle open structural, mechanical and functional questions of TJ physiology and pathology.