Abstract Background Degraded extracellular matrix can stimulate the innate immune system via the Toll-Like Receptor-4 (TLR4). In the pancreas, syndecan-anchored heparan sulphate (HS) on the ductal epithelium can be cleaved off its protein cores by the proteases (trypsin and elastase) and potentially activate TLR4 signalling. Methods To investigate this signalling event, a low sulphated HS (500 μg/ml) was infused into the biliary-pancreatic duct of C57BL/6J wild-type mice. Phosphate buffered saline (PBS) and lipopolysaccharide (LPS) were used as negative and positive controls, respectively. Mice were sacrificed after 1, 3, 6, 9, and 48 hours and tissues were analysed for neutrophil and cytokine contents. In order to study the TLR4 signalling pathway of HS in the pancreas, genetically engineered mice lacking TLR4, Myeloid Differentiation primary response gene (88) (MyD88) or Interferon Regulatory Factor 3 (IRF3) were subjected to pancreatic infusion of HS. Results Neutrophil sequestration and corresponding myeloperoxidase (MPO) activity in the pancreas were increased 9 hours following HS challenge. In wild-type mice, the monocyte chemoattractant protein-1(MCP-1) increased at 3 hours after infusion, while RANTES increased after 9 hours. TLR4, MyD88, and IRF3 knockout mice showed an abrogated neutrophil recruitment and myeloperoxidase activity in the HS group, while the LPS response was only abolished in TLR4 and MyD88 knockouts. Conclusions The results of this study show that HS is capable of initiating a TLR4-dependent innate immune response in the pancreas which is distinctly different from that induced by LPS. This inflammatory response was mediated predominantly through IRF3- dependent pathway. Release of HS into the pancreatic duct may be one important mediator in the pancreatic ductal defence.