ABSTRACT The human pathogens enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC), as well as the related mouse pathogen Citrobacter rodentium , utilize a type III secretion system (T3SS) to inject multiple effector proteins into host cells. The E. coli O157:H7 strain EDL933 carries two copies of non-locus of enterocyte effacement (LEE)-encoded protein H, designated NleH1 and NleH2, both of which bind to the human ribosomal protein S3 (RPS3), a subunit of NF-κB transcriptional complexes. In this study, we describe significant functional differences between NleH1 and NleH2 in their ability to regulate the host NF-κB pathway. We show that the EHEC and EPEC NleH effectors are functionally equivalent in their ability to affect RPS3 nuclear translocation. NleH1, but not NleH2, inhibited NF-κB activity without altering the kinetics of IκBα phosphorylation/degradation. We also determined that the class I PSD-95/Disc Large/ZO-1 (PDZ)-binding domain of NleH was important for its activity in the NF-κB pathway. In addition to binding RPS3, we found that NleH1 and NleH2 are able to bind to each other in vitro and in vivo , suggesting an additional mechanism by which the E. coli NleH effectors may regulate the extent and duration of NF-κB activation after their T3SS-dependent translocation. We also performed mouse infection experiments and established that mouse mortality and Citrobacter colonization were reduced in mice infected with Δ nleH . Complementing Δ nleH with NleH1 restored Citrobacter virulence and colonization to wild-type levels, whereas complementing with NleH2 reduced them. Taken together, our data show that NleH1 and NleH2 have pronounced functional differences in their ability to alter host transcriptional responses to bacterial infection.