Introduction: Researchers have investigated the potential role of intestinal permeability in Crohn's disease pathogenesis. Intestinal permeability is usually mediated by cytoskeleton and intercellular junctions. The myosin light chain kinase (MLCK) is an enzyme that activates the myosin light chain to exert its function related to cytoskeleton contraction and tight junction regulation. The correlation between MLCK and Crohn's disease pathogenesis has been consistently proven. Areas covered: This study aims to expand the understanding of the regulation and function of MLCK in Crohn's disease. An extensive literature search in the MEDLINE database (via PubMed) has been performed up to Oct. 2020. The roles of MLCK in tight junction activation, intestinal permeability enhancement, and cell signal regulation are comprehensively discussed. Expert opinion: Targeting the MLCK-related pathways such as TNF-α in CD treatment has been put into clinical use. More accurate targeting such as MLCK and TNFR2 has been proposed to reduce side effects. MLCK may also have the potential to become biomarkers in fields like CD activity. With the application of cutting age research methods and tools, the MLCK research could be accelerated.