Glucocorticoids, like prednisolone, are often used in clinic because of their anti-inflammatory and immunosuppressive properties. However, glucocorticoids reduce bone mineral density (BMD) as a side-effect. Malabsorption of Ca²⁺ in the intestine is supposed to play an important role in the aetiology of low BMD. To elucidate the mechanism of glucocorticoid-induced Ca²⁺ malabsorption, the present study investigated the effect of prednisolone on the expression and activity of proteins responsible for active intestinal Ca²⁺ absorption including the epithelial Ca²⁺ channel TRPV6, calbindin-D_9K and the plasma membrane ATPase PMCA1b. Therefore, C57BL/6 mice received 10 mg/kg bwt prednisolone daily by oral gavage for 7 days and were compared with control mice receiving vehicle only. An in vivo ⁴⁵Ca²⁺ absorption assay indicated that intestinal Ca²⁺ absorption was diminished after prednisolone treatment. We showed decreased duodenal TRPV6 and calbindin-D_9K mRNA and protein abundance in prednisolone-treated compared to control mice, whereas PMCA1b mRNA levels were not altered. Importantly, detailed expression studies demonstrated that in mice these Ca²⁺ transport proteins are predominantly localized in the first 2 cm of the duodenum. Furthermore, serum Ca²⁺ and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) concentrations remained unchanged by prednisolone treatment. In conclusion, these data suggest that prednisolone reduces the intestinal Ca²⁺ absorption capacity through diminished duodenal expression of the active Ca²⁺ transporters TRPV6 and calbindin-D_9K independent of systemic 1,25(OH)₂D₃.