Deregulation of c-myc expression is implicated in the pathogenesis of many neoplasias. Estrogen receptor alpha (ERalpha) can increase the rate of c-myc transcription through the recruitment of a variety of cofactors to the promoter, yet the precise roles of these cofactors in transcription and tumorigenesis are largely unknown. We show here that a putative tumor suppressor TIP30, also called CC3 or Htatip2, interacts with an ERalpha-interacting coactivator CIA. Using chromatin immunoprecipitation assays, we demonstrate that TIP30 and CIA are distinct cofactors that are dynamically associated with the promoter and downstream regions of the c-myc gene in response to estrogen. Both TIP30 and CIA are recruited to the c-myc gene promoter by liganded ERalpha in the second transcription cycle. TIP30 overexpression represses ERalpha-mediated c-myc transcription, whereas TIP30 deficiency enhances c-myc transcription in both the absence and presence of estrogen. Ectopic CIA cooperates with TIP30 to repress ERalpha-mediated c-myc transcription. Moreover, virgin TIP30 knockout mice exhibit increased c-myc expression in mammary glands. Together, these results reveal an important role for TIP30 in the regulation of ERalpha-mediated c-myc transcription and suggest a mechanism for tumorigenesis promoted by TIP30 deficiency.