Estrogen and progesterone, acting through their respective receptors and other nuclear proteins, exhibit opposing activities in target cells. We previously reported that Krüppel-like factor 9 (KLF9) cooperates with progesterone receptor (PR) to facilitate P-dependent gene transcription in uterine epithelial cells. Here we evaluated whether KLF9 may further support PR function by directly opposing estrogen receptor (ER) signaling. Using human Ishikawa endometrial epithelial cells, we showed that 17beta-estradiol (E(2))-dependent down-regulation of ERalpha expression was reversed by a small interfering RNA to KLF9. Transcription assays with the E(2)-sensitive 4x estrogen-responsive element-thymidine kinase-promoter-luciferase reporter gene demonstrated inhibition of ligand-dependent ERalpha transactivation with ectopic KLF9 expression. E(2) induced PR-A/B and PR-B isoform expression in the absence of effects on KLF9 levels. Addition of KLF9 small interfering RNA augmented E(2) induction of PR-A/B while abrogating that of PR-B, indicating selective E(2)-mediated inhibition of PR-A by KLF9. Chromatin immunoprecipitation of the ERalpha minimal promoter demonstrated KLF9 promotion of E(2)-dependent ERalpha association to a region containing functional GC-rich motifs. KLF9 inhibited the recruitment of the ERalpha coactivator specificity protein 1 (Sp1) to the PR proximal promoter region containing a half-estrogen responsive element and GC-rich sites, but had no effect on Sp1 association to the PR distal promoter region containing GC-rich sequences. In vivo association of KLF9 and Sp1, but not of ERalpha with KLF9 or Sp1, was observed in control and E(2)-treated cells. Our data identify KLF9 as a transcriptional repressor of ERalpha signaling and suggest that it may function at the node of PR and ER genomic pathways to influence cell proliferation.