The IL-1β-induced increase in intestinal epithelial tight junction (TJ) permeability has been postulated to be an important mechanism contributing to intestinal inflammation of Crohn’s disease and other inflammatory conditions of the gut. The intracellular and molecular mechanisms that mediate the IL-1β-induced increase in intestinal TJ permeability remain unclear. The purpose of this study was to elucidate the mechanisms that mediate the IL-1β-induced increase in intestinal TJ permeability. Specifically, the role of myosin L chain kinase (MLCK) was investigated. IL-1β caused a progressive increase in MLCK protein expression. The time course of IL-1β-induced increase in MLCK level correlated linearly with increase in Caco-2 TJ permeability. Inhibition of the IL-1β-induced increase in MLCK protein expression prevented the increase in Caco-2 TJ permeability. Inhibition of the IL-1β-induced increase in MLCK activity also prevented the increase in Caco-2 TJ permeability. Additionally, knock-down of MLCK protein expression by small interference RNA prevented the IL-1β-induced increase in Caco-2 TJ permeability. The IL-1β-induced increase in MLCK protein expression was preceded by an increase in MLCK mRNA expression. The IL-1β-induced increase in MLCK mRNA transcription and subsequent increase in MLCK protein expression and Caco-2 TJ permeability was mediated by activation of NF-κB. In conclusion, our data indicate that the IL-1β increase in Caco-2 TJ permeability was mediated by an increase in MLCK expression and activity. Our findings also indicate that the IL-1β-induced increase in MLCK protein expression and Caco-2 TJ permeability was mediated by an NF-κB-dependent increase in MLCK gene transcription.