Mast cells (MCs) have gained notoriety based on their detrimental pro-inflammatory contributions to IgE-mediated allergic disorders, such as asthma and life-threatening anaphylaxis. We have determined that the pro-inflammatory properties of MCs can be reduced by application of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the biologically active form of vitamin D3 (VitD3). A number of immune cell types have the ability to produce endogenous 1a25(OH)2D3 from the predominant inactive VitD3 metabolite in circulation, 25-hydroxyvitamin D3 (25OHD3), due to expression and activity of the hydroxylase enzyme CYP27B1. In this study, we explored if 25OHD3 can modulate IgE-dependent MC activation. Our data show, for the first time, that MCs express intracellular CYP27B1 and that 25OHD3 can be converted to significant levels of 1α,25(OH)2D3 by MCs. In addition, MC-CYP27B1 activity is required for 25OHD3 –induced inhibition of IgE-mediated MC-TNF and IL-6 production in vitro. By employing a mouse IgE-mediated MC-dependent passive cutaneous anaphylaxis (PCA) model, as well as four mouse groups with different cutaneous MC profiles in the ears, including WT mice, MC-deficient C57BL/6-KitW-sh/W-shmice and C57BL/6-KitW-sh/W-shmice engrafted with either WT or CYP27B1-/- MCs, we found that topical 25OHD3 application significantly reduced the magnitude of PCA-associated ear swelling in the WT MC engrafted group but not in mice that received CYP27B1-/- MCs. Taken together, these findings provide evidence that MCs are capable of utilising 25OHD3 to generate biologically active 1α,25(OH)2D3, and thereby extends the modus operandi of VitD3 (via both 25OHD3 and 1α,25(OH)2D3 metabolites) to efficiently negatively regulate IgE-mediated MC activation.