Giardia lamblia, a pathogen causing diarrhoeal outbreaks, is interesting how it triggers immune response in the human epithelial cells. This study defined the crucial roles of signalling components involved in G. lamblia-induced cytokine production in human epithelial cells. Incubation of the gastrointestinal cell line HT-29 with G. lamblia GS trophozoites triggered production of interleukin (IL)-1β, IL-8 and tumour necrosis factor (TNF)-α. IL-8 production was not significantly decreased by physically separating the HT-29 cells and G. lamblia GS trophozoites. Indeed, treatment of HT-29 with G. lamblia excretory–secretory products (ESP) induced IL-8 production. Electrophoretic mobility gel shift and transfection assays using mutagenized IL-8 promoter reporter plasmids indicated that IL-8 production by G. lamblia ESP occurs through activation of two transcriptional factors, nuclear factor kappaB (NF-κB) and activator protein 1 (AP-1) in HT-29 cells. In addition, activation of two mitogen-activated protein kinases (MAPKs), p38 and ERK1/2, was also detected in the HT-29 cells stimulated with G. lamblia ESP. Selective inhibition of these MAPKs resulted in decreased production of ESP-induced IL-8. These results indicate that activation of p38, ERK1/2 MAPK, NF-κB and AP-1 comprises the signalling pathway responsible for IL-8 production by G. lamblia ESP.