Enteropathogenic Escherichia coli (EPEC) blocks inflammatory signaling via a T3SS-dependent mechanism and recent research has shown that the effector protein NleE plays a key role in the inhibition of NF-κB activation. NleE inactivates the ubiquitin-chain binding activity of the host proteins TAB2 and TAB3 by modifying the NZF domain through S-adenosyl methionine (SAM) dependent cysteine methylation. TAB2 and TAB3 are homologous proteins with redundant functions that bind to TAK1 and are essential for NF-κB signaling. Previously, we identified the C-terminal motif 209IDSYMK241 of NleE as essential for inhibition of NF-κB activation. Here we determined that the 209IDSYMK241 motif of NleE was not involved in the recognition and binding of TAB3. Using yeast two hybrid protein interaction studies, we found that NleE6A , where 209IDSYMK241 was replaced with six alanine residues, retained the ability to bind TAB3. In contrast, the region between amino acids 34 to 52 of NleE, especially the motif 49GITR52, was critical for TAB3 κbinding. NleE mutants lacking the fragment between amino acids 34 and 52 had no inhibitory effect on NF-κB-dependent luciferase activity and during infection, wild-type NleE but not NleE49AAAA52 restored the ability of an nleE mutant to inhibit IL-8 production. In addition, ectopic expression of the NleE fragment from amino acids 34 to 52 (NleE34-52) in HeLa cells blocked the ability of wild-type NleE to suppress IL-8 secretion during EPEC infection. Lastly, we found that the CUE domain of TAB3 rather than the NZF domain was essential to maintain interactions with NleE. In summary, this work identified a unique TAB3 binding site in NleE and suggests that the sites of NleE and TAB3 interaction are distinct from the catalytic and modification sites respectively. However, all these regions are required for the ability of NleE to inhibit the host inflammatory response.