Listeriolysin O (LLO) is a pore-forming cytolysin secreted by the pathogen Listeria monocytogenes and is required for its intracellular survival. We recently demonstrated that in endothelial cells, LLO activates the NF-kappaB signalling pathway. In this work, we studied the LLO-induced molecular cascade of NF-kappaB activation with a cellular model extensively used to analyse the signalling pathway of NF-kappaB activation, i.e. the human embryonic kidney HEK-293 cell line and its derivatives (transfectants or mutants). When the stably transfected derivative HEK-293 cells expressing IL-1RI were exposed to LLO, a strong NF-kappaB activation was detected, contrasting with other cell lines (HEK-293 wild type, HEK-293.T and COS) expressing a very low level of IL-1RI. Although a delayed kinetics of LLO-dependent NF-kappaB activation suggests an autocrine or paracrine IL-1-dependent pathway, we found that LLO-dependent NF-kappaB activation did not require the IL-1 protein synthesis nor the interaction with the IL-1RI specific receptor. Herein, we demonstrated that LLO-dependent NF-kappaB activation requires the activation of the IkappaB kinase beta (IKKbeta) subunit of IKK complex to phosphorylate and degrade cytoplasmic IkappaBalpha, a natural inhibitor of NF-kappaB. The activation induced by LLO does not require the adapters MyD88 and IL-1R-associated kinase (IRAK). We suggested that LLO induces a distinct signalling pathway from that of IL-1 and its receptor.