Mitochondrial DNA (mtDNA) mutations have been described in almost all types of cancer. However, their exact role and timing of occurrence during tumor development and progression are still a matter of debate. A Vogelstein-like model of progression is well established for endometrial carcinoma (EC), however, mtDNA has been scarcely investigated in these tumors despite the fact that mitochondrial biogenesis increase has been shown to be a hallmark of type I EC. Here, we screened a panel of 23 type I EC tissues and matched typical hyperplasia for mutations in mtDNA and in four oncosupressors/oncogenes, namely PTEN, KRAS, CTNNB1 and TP53. Overall, mtDNA mutations were identified in 69% of cases, while mutational events in nuclear genes occurred in 56% of the cases, indicating that mtDNA mutations may precede the genetic instability of these genes canonically involved in progression from hyperplasia to tumor. Protein expression analysis revealed an increase in mitochondrial biogenesis and activation of oxidative stress response mechanisms in tumor tissues, but not in hyperplasia, in correlation with the occurrence of pathogenic mtDNA mutations. Our results point out an involvement of mtDNA mutations in EC progression and explain the increase in mitochondrial biogenesis of type I EC. Last, since mtDNA mutations occur after hyperplasia, their potential role in contributing to genetic instability may be envisioned.