Updated project metadata. Clustering of the Enteropathogenic (EPEC) Escherichia coli Tir effector, induced by its binding to Intimin, leads to pyroptotic cell death in macrophages. The effect of Tir clustering following EPEC infection of epithelial cells remains unexplored. In this study, we show that EPEC induces pyroptosis in an intestinal epithelial cell (IEC) line, in a Tir-dependent but actin polymerisation-independent manner, which was enhanced by priming with IFNγ. Mechanistically, Tir clustering induces rapid Ca2+ influx, which promotes internalisation of LPS, followed by activation of caspase-4. Chelation of extracellular Ca2+ or knockdown of caspase-4 inhibited cell death upon EPEC infection, whereas ATP-induced extracellular Ca2+ influx had the opposite effect confirming the regulatory role of calcium in the pathway. Additionally, IEC lines with low endogenous expression of caspase-4 were resistant to EPEC-induced cell death. We reveal a novel mechanism of LPS internalisation, following infection with an extracellular pathogen, leading to pyroptosis in IECs.