Calbindin-D(9k) (CaBP-9k) is a cytosolic calcium-binding protein mainly expressed in the duodenum, uterus and placenta, however, the role of CaBP-9k in the regulation of fetal growth remains to be elucidated. The present study was performed to investigate the expression pattern and regulation of CaBP-9k by antagonists of steroid hormones related with steroid hormone receptors during mid- and late pregnancy in mouse placenta and extra-embryonic membrane. The expression level of CaBP-9k increased in the placenta, while it decreased in the extra-embryonic membrane during pregnancy. The mRNA expression levels of estrogen receptor alpha (ERalpha) and progesterone receptor (PR) appeared to increase in both placenta and extra-embryonic membrane during pregnancy, suggesting that the ER and PR mRNA and protein expressions of placental CaBP-9k are positively correlated, but expressions of extra-embryonic membrane CaBP-9k are reversely correlated with ERalpha and PR mRNA levels. In addition, the present study indicates that the expressions of CaBP-9k mRNA and protein are differentially up- or down-regulated by antagonists of estrogen (E2) and progesterone (P4) in mouse placenta and extra-embryonic membranes, which suggests that E2 and P4 may be dominant factors in the regulation of the CaBP-9k. In particular, RU486, an antagonist of P4, down-regulated the mRNA and protein levels of placental CaBP-9k, whereas it up-regulated the protein level of extra-embryonic membrane CaBP-9k. In conclusion, we demonstrated that the CaBP-9k is distinctly regulated in the mouse placenta and extra-embryonic membrane, probably via sex steroid hormones (E2 and P4) and their receptors through a complex pathway. Extended studies are needed to verify relevant factors to regulate CaBP-9k gene and to provide further insight into roles of CaBP-9k gene in these tissues for the control of reproductive functions.