Integrin-linked kinase (ILK) is a ubiquitously expressed and highly conserved serine-threonine protein kinase that regulates cellular responses to a wide variety of extracellular stimuli. ILK is involved in cell-matrix interactions, cytoskeletal organization, and cell signaling. ILK signaling has also been implicated in oncogenesis and progression of cancers. However, its role in the innate immune system remains unknown. Here, we show that ILK mediates pro-inflammatory signaling in response to lipopolysaccharide (LPS). Pharmacological or genetic inhibition of ILK in mouse embryonic fibroblasts and macrophages selectively blocks LPS-induced production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha). ILK is required for LPS-induced activation of nuclear factor kappaB (NF-kappaB) and transcriptional induction of TNF-alpha. The modulation of LPS-induced TNF-alpha synthesis by ILK does not involve the classical NF-kappaB pathway, because IkappaB-alpha degradation and p65 nuclear translocation are both unaffected by ILK inhibition. Instead, ILK is involved in an alternative activation of NF-kappaB signaling by modulating the phosphorylation of p65 at Ser-536. Furthermore, ILK-mediated alternative NF-kappaB activation through p65 Ser-536 phosphorylation also occurs during Helicobacter pylori infection in macrophages and gastric cancer cells. Moreover, ILK is required for H. pylori-induced TNF-alpha secretion in macrophages. Although ILK-mediated phosphorylation of p65 at Ser-536 is independent of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway during LPS stimulation, upon H. pylori infection this event is dependent on the PI3K/Akt pathway. Our findings implicate ILK as a critical regulatory molecule for the NF-kappaB-mediated pro-inflammatory signaling pathway, which is essential for innate immune responses against pathogenic microorganisms.