Icb-1 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. To further elucidate the function of the icb-1 gene in differentiation of breast and endometrial cancer cells, we knocked down its expression by means of shRNA transfection. Knockdown of icb-1 inhibited the vitamin D3-induced up-regulation of E-cadherin expression in both MCF-7 and HEC-1B cells. Induction of E-cadherin expression by all-trans retinoic acid (ATRA) was also blocked in both cell lines expressing icb-1 siRNA. Examination of icb-1 and E-cadherin expression in 66 breast cancer tissue samples revealed a significant positive correlation between the two genes. In MCF-7 cells, silencing of the icb-1 gene inhibited the ATRA- and the vitamin D3-induced up-regulation of lactoferrin and estrogen receptor β expression. The data of our knockdown study suggest that icb-1 may act as a mediator of differentiation signals in breast cancer cells induced by ATRA or vitamin D3. These findings together with the observed co-expression of icb-1 with E-cadherin in breast cancer samples support an important role of the icb-1 gene in cancer cell differentiation.