Enteropathogenic Escherichia coli (EPEC), a major gastrointestinal pathogen, causes infantile diarrhea in many developing countries. EPEC is an attaching and effacing (A/E) pathogen that utilizes a LEE-encoded type III secretion system (TTSS) to deliver effector proteins into host cells. These effectors have been identified as potential virulence factors in A/E pathogens including EPEC, enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). We used a proteomics approach to identify a new non-LEE-encoded effector, NleI, from the EPEC sepL and sepD mutants. The nleI gene, located in a prophage-associated island with nleBCD, is also present in EHEC and CR but not in E. coli K-12. In EPEC, the transcription of nleI was increased upon sepD inactivation but remained unaffected in ler and sepL mutants. We demonstrated that NleI is secreted and translocated into HeLa cells in a TTSS-dependent manner and that the CesT chaperone is required for efficient NleI translocation. Nevertheless, under overexpression conditions, the first 20 amino acids of NleI are sufficient to support both secretion and translocation. After translocation, NleI can be detected in the cytoplasmic and membrane of HeLa cells.