p66 alpha is a GATA zinc finger domain-containing transcription factor that has been shown to be essential for gene silencing by participating in the NuRD complex. Several studies have suggested that p66 alpha is a risk gene for a wide spectrum of diseases such as diabetes, schizophrenia, and breast cancer; however, its biological role has not been defined. Here, we report that p66 alpha functions as a tumor suppressor to inhibit breast cancer cell growth and migration, evidenced by the fact that the depletion of p66 alpha results in accelerated tumor growth and migration of breast cancer cells. Mechanistically, immunoprecipitation assays identify p66 alpha as a p53-interacting protein that binds the DNA-binding domain of p53 molecule predominantly via its CR2 domain. Depletion of p66 alpha in multiple breast cells results in decreased expression of p53 target genes, while over-expression of p66 alpha results in increased expression of these target genes. Moreover, p66 alpha promotes the transactivity of p53 by enhancing p53 binding at target promoters. Together, these findings demonstrate that p66 alpha is a tumor suppressor by functioning as a co-activator of p53.