Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. Ca²⁺-mediated signal transduction pathways play a central regulatory role in DC responses to diverse antigens. 1,25-Dihydroxyvitamin D3 (1,25(OH)₂D₃) is a secosteroid hormone, that, in addition to its well-established action on Ca²⁺ homeostasis, possesses immunomodulatory properties. Although DCs are primary targets for 1,25(OH)₂D₃, nothing is known about its effects on DC Ca²⁺ channels and transporters. In the present study we show that mouse DCs incubated for 24 h with 1,25(OH)₂D₃ (100 nM) exhibit dramatically reduced Ca²⁺ entry upon acute application of lipopolysacharide (LPS, 100 ng/ml), an event known to be critical for DC maturation and function. We demonstrate that DCs express both, K⁺-independent (NCX1-3) and K⁺-dependent (NCKX1, 3-5), Na⁺/Ca²⁺ exchangers. Ca²⁺ imaging experiments and patch clamp current recordings revealed that at least one K⁺-dependent Na⁺/Ca²⁺ exchanger in DCs is upregulated by 1,25(OH)₂D₃. Moreover, 1,25(OH)₂D₃ upregulates the Ca²⁺-binding protein calbindin. Thus, decreased LPS-induced Ca²⁺ entry in 1,25(OH)₂D₃-incubated DCs can result from increased Ca²⁺ buffer capacity through calbindin and increased Ca²⁺ extrusion through Na⁺/Ca²⁺ exchanger.