The pathogenesis of endometriosis remains unclear, and relatively little is known about the mechanisms that promote establishment and survival of the disease. Previously, we demonstrated that v-akt murine thymoma viral oncogene homolog (AKT) activity was increased in endometriosis tissues and cells from ovarian endometriomas and that this increase promoted cell survival as well as decreased levels of progesterone receptor. The objective of this study was to demonstrate a role for AKT in the establishment of ectopic lesions. First, a dose-dependent inhibition of AKT in stromal cells from human ovarian endometriomas (OSIS) as well as endometrial stromal cells from disease-free patients (ESC) with the allosteric AKT inhibitor MK-2206 was demonstrated by decreased levels of phosphorylated (p)(Ser473)-AKT. Levels of the AKT target protein, p(Ser256)-forkhead box O1 were increased in OSIS cells, which decreased with MK-2206 treatment, whereas levels of p(Ser9)-glycogen synthase kinase 3β did not change in response to MK-2206. Although MK-2206 decreased viability of both OSIS and ESC in a dose-dependent manner, proliferation of OSIS cells was differentially decreased significantly compared with ESC. Next, the role of hyperactive AKT in the establishment of ectopic lesions was studied using the bigenic, PR(cre/+)Pten(f/+) heterozygous mouse. Autologous implantation of uterine tissues was performed in these mice. After 4 weeks, an average of 4 ± 0.33 lesions per Pten(f/+) mouse and 7.5 ± 0.43 lesions in the PR(cre/+)Pten(f/+) mouse were found. Histological examination of the lesions showed endometrial tissue-like morphology, which was similar in both the Pten(f/+) and PR(cre/+)Pten(f/+) mice. Treatment of mice with MK-2206 resulted in a significantly decreased number of lesions established. Immunohistochemical staining of ectopic lesions revealed decreased p(Ser473)-AKT and the proliferation marker Ki67 from MK-2206-treated mice compared with vehicle-treated mice. Furthermore, levels of FOXO1 and progesterone receptor increased in lesions of mice receiving MK-2206. These results demonstrate that heightened AKT activity plays an active role in the establishment of ectopic endometrial tissues.