Plasma membrane Ca²⁺ ATPases (PMCAs) are a ubiquitous system for the expulsion of Ca²⁺ from eukaryotic cells. In tight monolayers of polarized MDCK cells representing a distal kidney tubule model, PMCAs are responsible for about 1/3 of the vectorial Ca²⁺ transport under resting conditions, with the remainder being provided by the Na⁺/Ca²⁺exchanger (NCX). Vitamin D₃ (VitD) is known to increase PMCA expression and activity in Ca²⁺ transporting tissues such as the intestine, as well as in osteoblasts and MDBK epithelial cells. We found that VitD upregulated the expression of the PMCAs (mainly PMCA4b) in MDCK cell lysates at the RNA and protein level in a time- and dose-dependent manner. Interestingly, VitD caused a decrease of the PMCAs in the apical plasma membrane fraction and a concomitant increase of the pumps in the basolateral membrane. Functional studies demonstrated that transcellular ⁴⁵Ca²⁺ flux from the apical to the basolateral compartment was significantly enhanced by VitD. These findings demonstrate that VitD is a positive regulator of the PMCAs in MDCK epithelial cells. The correlation of decreased apical/increased basolateral expression of the PMCAs with an increase in transcellular Ca²⁺ flux from the apical (urine) towards the basolateral (blood) compartment indicates the physiological relevance of VitD function in kidney tubular Ca²⁺ reabsorption.