Listeria monocytogenes causes a pro-inflammatory response on adhesion to macrophages. Upregulation of inflammation genes involves the transcription factor NF-κB. Several components of L. monocytogenes, including lipoteichoic acid (LTA), phospholipases and listeriolysin O (LLO), have since been shown to mediate NF-κB activation. Here, we report that purified recombinant InIB, but not internalin (InIA), is a potent activator of NF-κB in the mouse macrophage-like cell line J774. Expression of InIB in Listeria innocua enhances its ability to activate NF-κB, while deletion of InIB from L. monocytogenes marginally decreases its effect on NF-κB, possibly because of the presence of NF-κB activators such as LTA and LLO. The effect correlates with the rapid degradation of IκBα, a sustained degradation of IKBβ and increases in tumour necrosis factor alpha (TNF-α) and interleukin (IL) 6 production, two cytokines controlled by NF-κB. Using a series of anti-InIB monoclonal antibodies and domains of InIB, NF-κB activation was shown to be dependent upon the N-terminal 213-amino-acid leucine-rich repeat (LRR) domain of InIB, recently demonstrated to be responsible for InIB-mediated L. monocytogenes invasion and phosphoinositide-3 (PI-3) kinase activation. The effect of InIB was blocked by PI-3 kinase inhibitors, indicating the involvement of PI-3 kinase in this response. This report thus illustrates that InIB not only promotes invasion, but also contributes to the macrophage pro-inflammatory response.