The orphan receptors, Rev-erbalpha and Rev-erbbeta, are members of the nuclear receptor superfamily and specifically repress apolipoprotein CIII (apoCIII) gene expression in rats and humans. Moreover, Rev-erbalpha null mutant mice have elevated very low density lipoprotein triacylglycerol and apoCIII levels. However, ligands for Rev-erb are unknown and the regulatory mechanism of Rev-erb is poorly understood. Conceivably, cofactors for Rev-erb may play an important role in the regulation of lipid metabolism. In this study, a zinc finger HIT domain-containing protein, ZNHIT-1, interacted with Rev-erbbeta. ZNHIT-1 was found to be a conserved protein in eukaryotes and was highly abundant in human liver. Furthermore, ZNHIT-1 was identified as a nuclear protein. Serial truncated fragments and substitution mutations established a putative nuclear localization signal at amino acids 38-47 of ZNHIT-1. A putative ligand-binding domain of Rev-erbbeta and the FxxLL motif of ZNHIT-1 were required for their interaction. Finally, ZNHIT-1 was recruited by Rev-erbbeta to the apoCIII promoter and removed the Rev-erbbeta-induced inhibition of apoCIII transcription. These findings demonstrate that ZNHIT-1 functions as a cofactor to regulate the activity of Rev-erbbeta, and may play a role in lipid metabolism.