Recent evidence shows that evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) interacts with tumor necrosis factor receptor–associated factor 6 (TRAF6), is ubiquitinated, and contributes to bactericidal activity during Toll-like receptor (TLR) signaling. Here we report a new regulatory role for ECSIT in TLR4 signaling. On TLR4 stimulation, endogenous ECSIT formed a molecular complex with p65/p50 NF-κB proteins. Our biochemical studies showed that ECSIT specifically interacted with p65/p50 NF-κB proteins, which colocalized in the nucleus. Of interest, these effects were critically dependent on ubiquitination of the ECSIT lysine (K) 372 residue. K372A mutant ECSIT did not interact with p65/p50 NF-κB proteins and markedly attenuated nuclear colocalization. In addition, ECSIT-knockdown THP-1 cells could not activate NF-κB DNA-binding activities of p65 and p50, production of proinflammatory cytokines, or NF-κB–dependent gene expression in response to TLR4 stimulation. However, these activities were markedly restored by expressing the wild-type ECSIT protein but not the K372A mutant ECSIT protein. These data strongly suggest that the ubiquitination of ECSIT might have a role in the regulation of NF-κB activity in TLR4 signaling.