Myeloid cells play a pivotal role in immune responses against bacterial and fungal infection. Among innate immune receptors, C-type lectin receptors (CLRs) can induce a wide spectrum of cytokines through immunoreceptor tyrosine-based activation motifs (ITAMs)-mediated signaling pathways. Dendritic cells (DCs) produce IL-10 through CLR stimulation; however, the regulatory mechanism of IL-10 expression has not been elucidated. In the current study, we report that calcium (Ca 2+) signaling-deficient DCs produced more IL-10 than wild type DCs. Mechanistically, Ca 2+-dependent phosphatase calcineurin directly inactivates cAMP response element binding protein (CREB), a transcription factor of Il10 in DCs, through dephosphorylating CREB at serine 133. In calcineurin-deficient DCs, CREB was highly phosphorylated and increased its binding to Il10 promoter. Elimination of MAPK signaling that phosphorylates CREB, deficiency of CREB, as well as deletion of CREB-binding site in Il10 promoter could diminish IL-10 production in DCs. Our findings identified a novel substrate of calcineurin as well as a mechanism through which Ca 2+ signaling regulates IL-10 expression downstream of CLRs. As IL-10 is a crucial immunosuppressive cytokine, this mechanism may counteract the over-activated IL-10 producing signals induced by CARD9 and MAPK pathways, preventing the ineffectiveness of immune system during bacterial and fungal infection.