Apurinic/apyrimidinic endonuclease 1 or redox factor-1 (Ape1/Ref-1) is a pleiotropic cellular protein involved in DNA repair and, through its redox activity, enhances the binding of a select group of transcription factors to their cognate recognition sequences in DNA. Thus, we were intrigued when we identified Ape1/Ref-1 and a number of DNA repair and oxidative stress proteins in a complex associated with the DNA-bound estrogen receptor alpha (ERalpha). Because Ape1/Ref-1 interacts with a number of transcription factors and influences their activity, we determined whether it might also influence ERalpha activity. We found that endogenously expressed Ape1/Ref-1 and ERalpha from MCF-7 human breast cancer cells interact and that Ape1/Ref-1 enhances the interaction of ERalpha with estrogen-response elements (EREs) in DNA. More importantly, Ape1/Ref-1 alters expression of the endogenous, estrogen-responsive progesterone receptor and pS2 genes in MCF-7 cells and associates with ERE-containing regions of these genes in native chromatin. Interestingly, knocking down Ape1/Ref-1 expression or inhibiting its redox activity with the small molecule inhibitor E3330 enhances estrogen responsiveness of the progesterone receptor and pS2 genes but does not alter the expression of the constitutively active 36B4 gene. Additionally, the reduced form of Ape1/Ref-1 increases and E3330 limits ERalpha-ERE complex formation in vitro and in native chromatin. Our studies demonstrate that Ape1/Ref-1 mediates its gene-specific effects, in part, by associating with endogenous, estrogen-responsive genes and that the redox activity of Ape1/Ref-1 is instrumental in altering estrogen-responsive gene expression.