ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor α (ERα) expression and a significant decrease of ERβ expression on the mRNA level. Expression of ERα-dependent genes progesterone receptor, pS2 , fibulin 1c , and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ERα transcript levels but sustaining high levels of ERβ, reducing both activation of ERα target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy.