In many type I endometrial cancers, the PTEN gene is inactivated which ultimately leads to constitutively active Akt and the inhibition of FOXO1, a member of the FOXO sub-family of Forkhead/winged helix family of transcription factors. The expression, regulation, and function of FOXO1 in endometrial cancer were investigated in this study. Immunohistochemical analysis of 49 endometrial tumor tissues revealed a decrease of FOXO1 expression in 95.9% of the cases compared to the expression in normal endometrium. In four different endometrial cancer cell lines (ECC1, Hec1B, Ishikawa, and RL95), FOXO1 mRNA was expressed at similar levels; however, protein levels were low or undetectable in Ecc1, Ishikawa and RL95 cells. Using siRNA technology, we demonstrated that the low levels of FOXO1 protein were due to the involvement of Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, given that silencing Skp2 increased FOXO1 protein expression in Ishikawa cells. Inhibition of Akt in Ishikawa cells also increased nuclear FOXO1 protein levels. Additionally, progestins increased FOXO1 protein levels, specifically through PRB as determined by using stably transfected PRA specific and PRB specific Ishikawa cell lines. Finally, overexpression of TmFOXO1 in the PR specific Ishikawa cell lines caused cell cycle arrest and significantly decreased proliferation in the presence and absence of the progestin, R5020. Furthermore, TmFOXO1 overexpression induced apoptosis in PRB specific cells in the presence and absence of ligand. Taken together, these data provide insight into the PI3K/Akt/FOXO pathway for the determination of progestin responsiveness and the development of alternate therapies for endometrial cancer.