Injury of the pancreatic duct epithelial barrier plays a critical role in the development of acute pancreatitis. The activity of the nuclear factor-kappa B (NF-κB) pathway is involved in the disruption of the pancreatic duct epithelial barrier. This study investigated how NF-κB impacts the dysfunction of the pancreatic duct epithelial barrier.A human pancreatic ductal adenocarcinoma cell line was treated with tumor necrosis factor-alpha (TNF-α) and pyrrolidine dithiocarbamate. The expression levels of p65 and p-p65 were detected to evaluate NF-κB activity. Tricellulin (TRIC) expression levels were measured to assess the change in tight junction (TJ)-related proteins. The expression and localization of myosin light chain kinase (MLCK) were investigated. The structure of TJs and monolayer permeability were also examined.NF-κB was activated by TNF-α and suppressed by pyrrolidine dithiocarbamate. Activation of NF-κB upregulated the expression levels of TRIC and MLCK. Broadened TJs were observed after NF-κB was activated. Lower monolayer permeability was observed when NF-κB was suppressed.Activation of the NF-κB pathway induced by TNF-α leads to increased TRIC and MLCK expression, resulting in broadened TJs and high permeability, which contribute to damage to the pancreatic duct epithelial barrier.