The type III secretion system (T3SS) effector protein NleE from enteropathogenic E. coli (EPEC) 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. Previously, we identified the C-terminal motif 209IDSYMK241 of NleE as essential for inhibition of NF-κB activation. 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 conserved region between amino acids 34 to 52 of NleE/OspZ, especially the motif 49GITR52, was critical for TAB3 binding. Further more, TAB2 and a newly identified NleE target, the NZF domain protein ZRANB3, were also associated with NleE/OspZ through the 49GITR52 motif. NleE mutants lacking 49GITR52 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. Here we also found that the homologue of NleE from Shigella flexneri 6, termed OspZ, bound TAB3 and decreased IL-8 transcription. In HT-29 cells, IL-8 expression levels were higher in cells infected with an ospZ mutant compared to wild type S. flexneri. Lastly, ectopic expression of an N-terminal fragment of NleE (NleE34-52)in HeLa cellsblocked the ability of wild-type NleE to suppress IL-8 secretion during EPEC infection. In summary, we have identified a unique substrate-binding motif in NleE and OspZ, which is required for the ability of NleE/OspZ to inhibit the host inflammatory response.