As a member of the family of potassium-dependent sodium/calcium exchangers, a sodium/calcium/potassium exchanger, NCKX3, plays a critical role in transport of 1 intracellular calcium and potassium ion in exchange for 4 extracellular sodium ions. Transcripts of NCKX3 were most abundant in the brain and smooth muscle, but in the uterus, aorta, and intestine, this gene expressed at lower levels. Its expression and role in the rat uterus during the estrus cycle have not been elucidated yet. In this study, we further examined the uterine expressions of NCKX3 mRNA and protein in stages during the estrous cycle of mature female Sprague-Dawley (SD) rats and in the absence or presence of sex-steroid hormones estrogen (E2) and progesterone (P4) in immature female SD rats. NCKX3 mRNA was verified by RT-PCR. Western blot analysis was applied to detect NCKX3 with an anti-NCKX3 goat polyclonal antibody. During the rat estrous cycle, uterus expressions of NCKX3 mRNA and proteins were highly expressed 2.5-fold at proestrus compared with those at estrus and diestrus. To examine the role(s) of sex steroids on the regulation of NCKX3 in the uterus of the immature female rat, hormones were prepared in ethanol and the rats were treated with E2 [40 (μg/kg of body weight (BW)], P4 (4 mg/kg of BW), or E2 plus P4 for 3 days. The expression of NCKX3 mRNA and protein increased 2-fold with E2, whereas P4 antagonized E2-stimulated NCKX3 expression, as similarly observed in the uterus. In addition, spatial expression of NCKX3 protein was detected by immunohistochemistry. Uterine NCKX3 protein was abundantly localized in the cytoplasm of the luminal and glandular epithelial cells at proestrus. Taken together, these results indicate that uterine NCKX3 is abundantly expressed in the uterus and controlled by steroid hormones E2 and P4, suggesting that uterine expression of NCKX3 might be involved in reproductive function during the cycle in female rat.