AP-2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP-2alpha-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-2alpha 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-2alpha-mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1-mediated repression of AP-2alpha transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP-2alpha, suggesting that KCTD1 specifically acts as a negative regulator of AP-2alpha. 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-2alpha.