Insulin-like growth factor-I receptor signaling contributes to the development of endometrial hyperplasia, the precursor to endometrioid-type endometrial carcinoma, in humans and in rodent models. This pathway is under both positive and negative regulation, including S6 kinase (S6K) phosphorylation of insulin receptor substrate-1 (IRS-1) at S636/639, which occurs downstream of mammalian target of rapamycin (mTOR) activation to inhibit this adapter protein. We observed activation of mTOR with a high frequency in human endometrial hyperplasia and carcinoma, but an absence of IRS-1 phosphorylation, despite high levels of activated S6K. To explore when during disease progression mammalian target of rapamycin (mTOR) activation and loss of negative feedback to IRS-1 occurred, we used the Eker rat (Tsc2^Ek/+) model, where endometrial hyperplasia develops as a result of loss of Tsc2, a “gatekeeper” for mTOR. We observed mTOR activation early in progression in hyperplasias and in some histologically normal epithelial cells, suggesting that event(s) in addition to loss of Tsc2 were required for progression to hyperplasia. In contrast, whereas IRS-1 S636/639 phosphorylation was observed in normal epithelium, it was absent from all hyperplasias, indicating loss of IRS-1 inhibition by S6K occurred during progression to hyperplasia. Treatment with a mTOR inhibitor (WAY-129327) significantly decreased hyperplasia incidence and proliferative indices. Because progression from normal epithelium to carcinoma proceeds through endometrial hyperplasia, these data suggest a progression sequence where activation of mTOR is followed by loss of negative feedback to IRS-1 during the initial stages of development of this disease. Cancer Prev Res; 3(3); 290–300