Previously, we investigated the induction effect of LRP16 expression by estrogen (17β-estradiol, E2) and established a feed-forward mechanism that activated estrogen receptor α (ERα) transactivation in estrogen-dependent epithelial cancer cells. LRP16 is required for ERα signaling transduction by functioning as an ERα coactivator. In this study, we demonstrated that LRP16 expression was upregulated in E2-responsive BG-1 ovarian cancer cells, but was downregulated in estrogen-resistant SKOV3 ovarian cancer cells. Pure estrogen antagonist ICI 182 780 did not affect LRP16 expression in SKOV3 cell. The unliganded ERα upregulated LRP16 expression and enhanced LRP16 promoter activity in SKOV3 cells; however, this induction was blocked by estrogen stimulation. Results from chromatin immunoprecipitation experiment revealed a strong recruitment of the unliganded ERα at LRP16 promoter in the absence of estrogen; however, ERα was largely released from the DNA upon E2 stimulation. Modulation in LRP16 expression level did not significantly change the proliferation rate of SKOV3 cells and the growth responsiveness of cells to E2. Knockdown of LRP16 by RNA interference in SKOV3 cells markedly attenuated estrogen response element-dependent ERα reporter gene activity and E2-induced c-Myc expression. Our study suggests a novel mechanism of estrogen resistance of ovarian cancer by which estrogen-repressed signaling pathway antagonizes estrogen-activated signaling transduction.