Toll-like receptors (TLRs) play an important role as a sensor of microbial pathogens in the innate immune response. TLRs transmit signals through the recruitment of adaptor proteins including tumor necrosis factor-associated factor 6 (TRAF6). which mediates the activation of I kappa B kinase (IKK). TIFA (TRAF-interacting protein with a forkhead-associated (FHA) domain) has been shown to bind to TRAF6 and activate IKK by promoting the oligomerization and Ubiquitin-ligase activity of TRAF6. FHA domains preferentially bind to phospho-threonine residues in their targets. Here. we identified a novel zinc finger protein, ZCCHC 11, that interacts with TIFA from phosphoproteins of a macrophage cell line. RAW 264.7, by Using affinity purification with GST-TIFA and mass spectrometric analysis. By a search of the EST database. we found a 200 kDa full-length form (ZCCHC11L). ZCCHC11L\vas mostly located to the nucleus, but translocated into the cytoplasm in response to LPS and bound to TIFA. Overexpression and knockdown by siRNA indicated that ZCCHC11 functions as a negative regulator of TLR-mediated NF-kappa B activation. The N-terminal region (ZCCHC11S) including CCHC-type Zn-finger motif was sufficient for Suppression of NF-kappa B. We propose that ZCCHC11 is a unique TLR signal regulator. which interacts with TIFA after LPS treatment and suppresses the TRAF6-dependent activation of NF-kappa B. (c) 2006 Elsevier Inc. All rights reserved.