AP‐2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP‐2α‐interacting partners, a yeast two‐hybrid screen was performed in human brain cDNA library. One of the identified clones encodes potassium channel tetramerization domain‐containing 1 (KCTD1). We demonstrated the novel KCTD1–AP‐2α interaction in vitro by GST pull‐down assays and in vivo by co‐immunoprecipitation assays and mapped the interaction domains to the N‐termini of both proteins. In addition, we observed that the two proteins were completely co‐localized in the nuclei of mammalian cells. Transient transfection assays using four promoters containing AP‐2‐binding sites confirmed that KCTD1 significantly repressed AP‐2α‐mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1‐mediated repression of AP‐2α transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP‐2α, suggesting that KCTD1 specifically acts as a negative regulator of AP‐2α. Finally, we found that KCTD1 interacted with three major members of the AP‐2 family and inhibited their transcriptional activities. Taken together, our results indicate the novel function of KCTD1 as the transcriptional repressor for AP‐2 family, especially for AP‐2α. J. Cell. Biochem. 106: 285–295, 2009. © 2008 Wiley‐Liss, Inc.