Estrogen has direct and indirect effects on mitochondrial activity, but the mechanisms mediating these effects remain unclear. Others reported that long term estradiol (E₂) treatment increased Nuclear Respiratory Factor-1 (NRF-1) protein in cerebral blood vessels of ovariectomized rats. NRF-1 is a transcription factor that regulates the expression of nuclear-encoded mitochondrial genes, e.g., mitochondrial transcription factor A (TFAM), that control transcription of the mitochondrial genome. Here we tested the hypothesis that E₂ increases NRF-1 transcription resulting in a coordinate increase in the expression of nuclear- and mitochondrial- encoded genes and mitochondrial respiratory activity. We show that E₂ increased NRF-1 mRNA and protein in MCF-7 breast and H1793 lung adenocarcinoma cells in a time-dependent manner. E₂-induced NRF-1 expression was inhibited by the estrogen receptor (ER) antagonist ICI 182,780 and Actinomycin D, but not by phosphoinositide-3 kinase and MAPK inhibitors, indicating a genomic mechanism of E₂ regulation of NRF-1 transcription. An estrogen response element (ERE) in the NRF-1 promoter bound ER and ER in vitro and E₂ induced ER and ER recruitment to this ERE in chromatin immunoprecipitation assays in MCF-7 cells. The NRF-1 ERE activated reporter gene expression in transfected cells. siRNA to ER and ER revealed that ER mediates E₂-induced NRF-1 transcription. The E₂-induced increase in NRF-1 was followed by increased TFAM and the transcription of Tfam-regulated mtDNA-encoded COI and NDI genes and increased mitochondrial biogenesis. Knockdown of NRF-1 blocked E₂-stimulation of mitochondrial biogenesis and activity indicating a mechanism by which estrogens regulate mitochondrial function by increasing NRF-1 expression.