Previously, we reported that renal active calcium transporting genes are highly expressed in female mice, and suggested that renal calcium processing genes play a critical role in normal calcium re-absorption in females. In the current study, we evaluated the differential expression of renal calcium processing genes in male and female mice. Using microarray analysis, we identified K⁺-dependent Na⁺/Ca²⁺ exchanger 3 (NCKX3) as a gene that was differentially expressed in the kidneys of female and male mice. The expression levels of renal NCKX3 mRNA and protein were higher in female than in male mice, whereas there was no difference between the genders in the levels of NCKX3 expression in the brain. Renal NCKX3 localized to the basolateral layer of distal convoluted tubules, indicating that this protein participates in renal calcium re-absorption. To identify putative regulators of the gender-specific expression of NCKX3, sex-steroid hormones were injected into mature female and male mice. Aldosterone and hydrocortisone from the adrenal gland down-regulated renal NCKX3 expression in female mice, whereas other sex steroids had no effect. The results of this study suggest that females maintain a high level of renal NCKX3 expression in distal convoluted tubules, and that the regulation of NCKX3 expression by adrenal gland-secreted hormones may play a role in active calcium transport in the kidneys of female mice.