Enteropathogenic E. coli (EPEC) is a gastrointestinal pathogen that adheres intimately to the apical surface of intestinal enterocytes and causes diarrhoea in infants, particularly in developing countries. EPEC utilises a type III secretion system (T3SS) to directly translocate a diverse repertoire of virulence (effector) proteins directly into host cells. The effector proteins subvert a multitude of host cellular processes including innate immune signaling and apoptosis in order to persist in the host. We have characterized the host targets for a number of these virulence effectors, and defined the physiological relevance of their activities. The host immune signaling proteins RIPK1, RIPK3, TRIF and ZBP1/DAI, all contain receptor-interacting protein (RIP) homotypic interaction motifs (RHIM), and play a key role in cell death and inflammatory signaling. RHIM-dependent interactions help drive a caspase-independent form of cell death termed necroptosis. Here we report that EPEC uses the T3SS effector EspL to directly cleave and inactivate RIPK1, RIPK3, TRIF and ZBP1/DAI during infection. This required a previously unrecognised tripartite cysteine protease motif in EspL (Cys⁴⁷, His¹³¹, Asp¹⁵³) that cleaved within the RHIM of these proteins. Bacterial infection and/or ectopic expression of EspL led to rapid inactivation of RIPK1, RIPK3, TRIF and ZBP1/DAI and inhibition of TNF, LPS or poly (I:C)-induced necroptosis and inflammatory signaling. Furthermore, EPEC infection inhibited TNF-induced phosphorylation and plasma membrane localization of MLKL. In vivo, Ripk3^-/- but not Mlkl^-/- mice exhibited increased pathology upon infection with the EPEC-like mouse pathogen Citrobacter rodentium compared to wild type C57BL/6 mice. The activity of EspL defines a new family of T3SS cysteine protease effectors found in a range of bacteria and reveals a new mechanism by which gastrointestinal pathogens directly target RHIM-dependent inflammatory and necroptotic signaling pathways.