Introduction: Optimal decidualization of the endometrium is critical for successful embryo implantation and placentation. In contrast to most mammalian viviparous species, decidualization in humans occurs spontaneously every menstrual cycle under the influence of steroid hormones in the absence of an embryo. Forkhead box protein O1 (FOXO1) is a master transcription factor that regulates decidualization in response to progesterone stimulation. We hypothesize that FOXO1 dysregulation leads to abnormal decidualization and subsequent pregnancy complications. We examined FOXO1 expression in tissues from patients with pregnancy complications (pre-eclampsia (PE), spontaneous preterm birth (sPTB)) and tested the function of FOXO1 by knocking down its expression in human endometrial stromal cells (HESCs) and measuring decidualization biomarkers in conditioned medium. Methods: Immunohistochemistry of FOXO1 expression was performed in fetal membranes/placentas collected from 10 term births, 11 PE, and 5 sPTB (26-34 weeks). Two approaches were used to knockdown FOXO1 function in HESCs: (1) CRISPR-Cas9 nucleoriboprotein complex transfection, and (2) transduction with lentivirus harboring short-hairpin RNA constructs. Prolactin and IGFBP1 ELISA was performed to test the function of knockdown HESCs after induction of decidualization by 1 μM of medroxyprogesterone acetate (MPA) and 0.5 mM of dibutyryl-cAMP for 12 days. Cell proliferation was measured by MTT assay. Results: Expression of FOXO1 was localized only to decidual cells in fetal membranes. Significant differences in FOXO1 expression were found from term birth, PE, and sPTB (P<0.001, Kruskal-Wallis test), with reduced expression of FOXO1 in both PE (P=0.002) and sPTB (P=0.006) versus term birth (Mann-Whitney analysis with Bonferroni correction). FOXO1 expression was significantly correlated with gestational length (r=0.767, P<0.001). HESCs with knockdown of FOXO1 did not show significant change in proliferation. However, there was a significant reduction in the production of the decidualization biomarkers, prolactin and IGFBP1. Conclusion: FOXO1 expression in decidual cells of PE and sPTB fetal membranes was significantly reduced compared with term birth membranes. Knockdown of FOXO1 in HESCs impaired the capacity of the cells to undergo decidualization. Taken together, these results suggest that FOXO1 is a critical regulator of endometrial decidualization and dysregulation of FOXO1 may be important in the mechanism of sPTB.