The nuclear transcription factor signal transducer and activator of transcription 3 (Stat3) has recently been reported to have a localized mitochondrial regulatory function. Current data suggest that mitochondrial Stat3 (mitoStat3) is necessary for maximal mitochondrial activity and for Ras-mediated transformation independent of Stat3 nuclear activity. We have previously shown that Stat3 has a pivotal role in epithelial carcinogenesis. Therefore, the aim of the current study was to determine the role of mitoStat3 in epidermal keratinocytes. Herein, we show that normal and neoplastic keratinocytes contain a pool of mitoStat3. EGF and 12-O-tetradecanoylphorbol-13-acetate induce Stat3 mitochondrial translocation mediated through the phosphorylation of Stat3 at Ser727. In addition, we report that mitoStat3 binds mtDNA and associates with the mitochondrial transcription factor A. Furthermore, Stat3 ablation resulted in an increase of mitochondrial-encoded gene transcripts. An increase in key nuclear-encoded metabolic genes, PGC-1α and NRF-1, was also observed in Stat3-null keratinocytes; however, no changes in nuclear-encoded electron transport chain gene transcripts or mtDNA copy number were observed. Collectively, our findings suggest a heretofore-unreported function for mitoStat3 as a potential mitochondrial transcription factor in keratinocytes. This mitoStat3-mtDNA interaction may represent an alternate signaling pathway that could alter mitochondrial function and biogenesis and have a role in tumorigenesis.