Although Myc-interacting zinc-finger protein-1 (Miz-1) is known to be a poxvirus and zinc-finger (POZ) transcription factor required for Myc transcriptional repression, additional regulatory function of Miz-1 is less well understood. Using a yeast two-hybrid screen, we identified human alternate reading frame (ARF) protein as a novel interaction partner of Miz-1. The zinc-finger domain of Miz-1 is involved in its binding to ARF. In addition, we found that Miz-1 was able to interact with p53 through its DNA-binding domain, thus to diminish the binding of p53 to its target promoter and inhibit p53-mediated gene transcription. Interestingly, the Miz-1-regulated p53 transcriptional suppression does not require the presence of ARF or Mdm2. Importantly, ARF and p53 were found to competitively bind to Miz-1 in regulating p53-mediated transcription, and this conclusion was verified by both in vitro binding assay and competitive chromatin immunoprecipitation assay using a bona fide p53 endogenous Bax and Puma promoters. Thus, our study reveals that Miz-1 acts as a p53 suppressor by interfering with p53 DNA-binding ability, and ARF is able to counteract the suppression of Miz-1 on p53 by direct binding to Miz-1, suggesting that Miz-1 is a novel mediator in the ARF-p53 pathway.