Background and aims: Helicobacter pullorum is an enterohepatic Helicobacter species of avian origin detected in patients with acute diarrhoea and inflammatory bowel disease. The aim of the present study was to determine whether H pullorum exerts a direct effect on human intestinal epithelial cells in vitro and to characterise the bacterial mechanisms and the signalling pathways involved. Materials and methods: The proinflammatory properties of H pullorum from human and avian origins were measured on human gastric (AGS) and intestinal (CaCo-2 and HT-29) epithelial cell lines after co-culture with different H pullorum strains, and the extent of nuclear factor-κB (NF-κB) involvement was determined. Results: All of the H pullorum strains tested stimulated interleukin 8 (IL8) secretion by the three cell lines. Similar results were obtained with heat-killed H pullorum. Incubation of cells with filtered H pullorum culture supernatants did not stimulate IL8 secretion. The same observation was made when bacterial adherence was inhibited by Transwell inserts. H pullorum induced NF-κB activation and rapid nuclear translocation as demonstrated by immunofluorescent staining and cellular fractionation. NF-κB involvement was confirmed by using the specific inhibitor SN50 and small interfering RNA (siRNA) which abolished H pullorum-induced IL8 production. Conclusions: H pullorum strains stimulate IL8 secretion by human gastric and intestinal epithelial cell lines. This effect requires bacterial adherence and probably lipopolysaccharides, and is mediated by NF-κB signalling. The present study strengthens the argument that H pullorum is a potent human pathogen and highlights its putative role in acute and chronic digestive diseases such as inflammatory bowel disease.