Peroxiredoxin 5 (Prx5) is a member of a family consisting of six antioxidant enzymes. Prx5 is ubiquitously expressed in various tissues including mitochondria and peroxisomes, implying that Prx5 functions as a regulator of the cellular oxidation state. Prx5 plays a critical role in protecting cells from oxidative stress by inhibiting the accumulation of reactive oxygen species and cell death. Overexpression of Prx5 in mammary tissue is associated with poor prognosis of breast cancer. The present study was conducted to elucidate the regulatory mechanisms of Prx5 gene expression and the physiological relevance of Prx5 in breast cancer cell survival. Analysis of the promoter region of the Prx5 gene and reporter gene assays revealed the promoter region critical for Prx5 gene regulation to which the novel negative transcription regulator, GATA1 binds in human breast cancer cell lines. GATA1 was further confirmed as a potential transcription factor for Prx5 gene expression in another set of transfection assays using a reporter gene construct that contained a mutated GATA1 binding site. Direct binding of GATA1 to the Prx5 promoter was demonstrated using chromatin immunoprecipitation and electrophoretic mobility shift assays. Finally, functions of Prx5 associated with apoptosis were examined in two human breast cancer cell lines displaying high levels of either Prx5 or GATA1 expression. Knockdown of GATA1 led to increased expression of Prx5 and inhibition of apoptosis. Our data suggest that Prx5 may protect cells from oxidative stress-mediated apoptosis in a GATA1-regulated manner.