Many bacterial pathogens transport virulence proteins, termed effector proteins, into host cells via specialized protein secretion systems such as type III secretion systems (T3SS). Enteropathogenic Escherichia coli (EPEC) blocks inflammatory signaling via a T3SS-dependent mechanism and recent research has shown that the effector protein NleE is necessary and sufficient to block NF-κB activation. NleE inactivates the ubiquitin-chain binding activity of the host proteins TAB2 and TAB3 by modifying the ZnF domain through SAM dependent cysteine methylation. The C-terminal motif 209IDSYMK241 of NleE is the suggested catalytic site that is essential for enzymatic function. TAB2 and TAB3 are homologous proteins with redundant functions that bind to TAK1 and are therefore involved in NF-κB signaling pathway. Using the Yeast Two Hybrid System, we found that NleE209AAAAAA214 retains the ability to bind TAB3. In contrast, the region between amino acid 34 to 52 of NleE, especially the motif 49GITR52, is critical for binding. In addition, we found that the CUE domain of TAB3 rather than the ZnF domain is essential for NleE interaction. NleE missing the fragment between amino acids 34 and 52 had no inhibitory effect on NF-κB-dependent luciferase activity. During infection, nleE mutant derivatives of EPEC showed a diminished capacity to inhibit IL-8 production, which was complemented to wild type levels upon the reintroduction of wild-type NleE but not NleE49AAAA52. The data presented here suggests that the sites of NleE and TAB3 interaction are different from the catalytic and modification sites of NleE and TAB3 respectively and that these regions are all required for the ability of NleE to inhibit the host immune response.