Increasing evidence suggests that the tight junction is a dynamically regulated structure. Cytoskeletal reorganization, particularly myosin light chain phosphorylation--induced actomyosin contraction, has increasingly been recognized as a mediator of physiological and pathophysiological tight junction regulation. However, our understanding of molecular mechanisms of tight junction modulation remains limited. Recent studies using live cell and live animal imaging techniques allowed us to peek into the molecular details of tight junction regulation. At resting conditions, the tight junction is maintained by dynamic protein–protein interactions, which may provide a platform for rapid tight junction regulation. Following stimulation, distinct forms of tight junction protein reorganization were observed. Tumor necrosis factor (TNF-α) causes a myosin light chain kinase (MLCK)--mediated barrier regulation by inducing occludin removal from the tight junction through caveolar endocytosis. In contrast, MLCK- and CK2-inhibition--caused tight junction regulation is mediated by altered zonula occludens (ZO)-1 protein dynamics and requires ZO-1--mediated protein–protein interaction, potentially through regulating claudin function. Although some of the molecular details are missing, studies summarized above point to modulating protein localization and dynamics that are common mechanisms for tight junction regulation.