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-Kit^W-sh/W-shmice and C57BL/6-Kit^W-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.