Zhangfei is a basic region-leucine zipper (bZIP) transcription factor identified through its interaction with a herpesvirus-related host cell factor HCF1 (C1). Unlike most bZIP proteins, the mammalian Zhangfei protein does not bind DNA as homodimers. It is believed due to the absence of an asparagine residue in the basic region, which forms the DNA-recognition motif, NxxAAxxCR, in all bZIP proteins. Here, we report the identification and characterization of a novel Zhangfei homologue in Takifugu rubripes, which has an intact DNA-recognition motif by sequence analysis. We found that the pufferfish Zhangfei (pZF) appeared to have all the functional domains known in human Zhangfei, including the conserved HCF1-binding motif; however, pZF did not appear to bind DNA either. These findings suggest that the distinct property of the Zhangfei basic region is conserved during the evolution of vertebrates and that Zhangfei requires interaction with other proteins to regulate transcription from target promoters.