Severe acute pancreatitis (SAP) often results in multiple-organ dysfunction syndrome with high mortality. There is no effective clinical therapy for SAP, yet daphnetin, a coumarin extracted from Dracaena marginata, has analgesic and anti-inflammatory effects, and has been used clinically in several diseases. The objective of this study was to investigate the role and underlying mechanisms of daphnetin in a rat SAP model. Male Wistar rats were pretreated with daphnetin via intraperitoneal injection, 30[Formula: see text]min before retrograde infusion of 5% sodium taurocholate into the biliopancreatic duct. Twelve hours after sodium taurocholate administration, rats were sacrificed and tissues and blood were harvested. Then, histological, chemical, and molecular analyses were performed. Daphnetin treatment reduced the levels of serum alanine transaminase and creatinine (CR), increased superoxide dismutase(SOD) activity, and decreased neutrophil infiltration and cell apoptosis of the pancreatic tissues in rat SAP. Daphnetin treatment significantly decreased expression of pro-inflammatory cytokines and increased expression of anti-inflammatory cytokines in rat SAP. Molecular analyses revealed that daphnetin reduced TLR4 expression and inhibited NF-[Formula: see text]B signaling pathway activation. These findings demonstrate that daphnetin attenuates acute pancreatic injury by regulating the TLR4/NF-[Formula: see text]B signaling pathway and inflammation in rat SAP model. Daphnetin may be a potential therapeutic agent for SAP.