Aims: Dendritic cells (DCs) are antigen-presenting cells that provide a link between innate and adaptive immunity. 1,25-Dihydroxyvitamin D3 (1,25(OH)₂D₃) and dexamethasone (Dex) are powerful immunosuppressors that strongly affect DC functions. On the other hand, DC functions are controlled by Ca²⁺ signaling. The present study explored whether 1,25(OH)₂D₃ and Dex influence cytosolic Ca²⁺ concentration in DCs. Methods: Whole-cell patch-clamp experiments and measurements of cytosolic Ca²⁺ concentration by Fura-2 fluorescence were performed and expression of maturation markers by DCs was analyzed by flow cytometry. Results: Here we provide evidence that both hormones upregulate Na⁺/Ca²⁺ exchangers in DCs. We further demonstrate that this effect results in reduced increase of cytosolic Ca²⁺ upon application of lipopolysacharide (LPS, 100 ng/ml), an event known to be critical for DC maturation and function. LPS-induced expression of the costimulatory molecule CD86 was down-regulated by 1,25(OH)₂D₃ and Dex, an effect significantly blunted by the Na⁺/Ca²⁺ exchanger blockers 3',4'-dichlorobenzamyl and KB-R7943. Conclusion: 1,25(OH)₂D₃ and Dex blunt the LPS-induced increase in cytosolic Ca²⁺ by stimulation of Na⁺/Ca²⁺ exchanger-dependent Ca²⁺ extrusion. The effect contributes to the signaling underlying the inhibition of DC maturation by these immunosuppressive hormones.