Dexamethasone (DEX) is a member of the glucocorticoids (GCs), and is broadly used as an anti-inflammatory medication. Continuous administration with GCs induces adverse effects and suffering in human (i.e., osteoporosis) due to negative calcium balance derived from low re- and absorption in the duodenum and kidney. A cytosolic calcium binding protein, Calbindin-D9k (CaBP-9k), is dominantly expressed in the renal and intestinal tissues involved in calcium re- and absorption and plays an active role in calcium transport. In the present study, we employed adrenalectomized (ADX) and Sham treated male mice to examine the effect of DEX on CaBP-9k gene expression in the duodenum and kidney. DEX significantly reduced the levels of duodenal CaBP-9k mRNA and protein, and it restored ADX-induced decrease in renal CaBP-9k protein, as compared to the level of Sham control. DEX treatment increased calcium and phosphate levels in the sera of both Sham and ADX mice. In a time-course experiment, DEX significantly decreased duodenal CaBP-9k at the transcriptional and translational levels at 3 days, while it temporarily increased CaBP-9k mRNA and protein levels at 12 and 24 h. Altered CaBP-9k expression by DEX was completely reversed by mifepristone, an antagonist for glucocorticoid receptor (GR). In addition, duodenal CaBP-9k and GR were co-localized on the enterocyte (duodenocyte), supporting a role for GR in regulating CaBP-9k. In ovariectomized (OVX) and ADX female mice daily treated DEX for 3 days, duodenal CaBP-9k was expressed same as its level in male mice. Also, no cross activity of progesterone and DEX on their receptors was observed. Taken together, these results indicate that mouse CaBP-9k gene may be regulated by DEX in a tissue-specific manner and reduced duodenal CaBP-9k via GR pathway may take part in negative calcium absorption of glucocorticoid-induced osteoporosis, while renal CaBP-9k may not be involve in the regulation of calcium homeostasis.