Programmed cell death pathways are crucial for maintaining homeostasis, eliminating damaged or diseased cells before they can cause harm. Thus, the understanding of the mechanisms involved in these pathways is necessary for future therapeutic development. Pyroptosis, a pro-inflammatory lytic form of cell death, restricts the growth of both pathogens and cancer cells and effectively stimulates immune responses. In this study I characterised the mechanism resulting in pyroptosis of colorectal cancer cell (CRC) lines infected by a tumour-colonising bacterium, enteropathogenic Escherichia coli (EPEC). EPEC uses a type III secretion system to inject virulence factors, called effectors, into the host cytosol. The translocated intimin receptor (Tir), an essential effector that inserts into the host plasma membrane and binds to the bacterial outer membrane protein intimin to establish the intimate attachment of the bacteria, activates pyroptosis in CRC cells. Pyroptosis is initiated by Tir-dependent extracellular Ca2+ influx through the mechanosensitive cation channel TRPV2, followed by the internalisation of the bacterial cell wall component lipopolysaccharide (LPS). Cytosolic LPS can be recognised by caspase-4, which then cleaves and activates the pore-forming protein gasdermin D to facilitate cell lysis. As pyroptosis and its downstream immune stimulation is detrimental to EPEC colonisation, multiple other bacterial effectors are employed to counteract pyroptosis at different stages. Importantly, this Tir-dependent cell death mechanism is largely conserved among the caspase-4- and GSDMD-expressing CRC cells of different genetic backgrounds. This study identifies a novel bacterial virulence factor-mediated pyroptotic mechanism effective in killing CRC cells that could potentially be exploited for future cancer therapy development.