Mast cells (MCs) are typically regarded as pro-inflammatory effector cells in immunoglobulin (Ig)E-dependent allergic disorders such as asthma and life-threatening anaphylaxis. However, we have determined that the pro-inflammatory properties of MCs can be restrained by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the biologically active form of vitamin D3 (VitD3)1. In this study we explored whether the predominant inactive form of VitD3 in circulation, 25-hydroxyvitamin D3 (25OHD3), can also modulate IgE-dependent MC activation; a function that would require MCs to endogenously convert 25OHD3 to (1α,25(OH)2D3). Our data show, for the first time, that MCs express the hydroxylase CYP27B1 and that 25OHD3 can be converted to significant levels of 1α,25(OH)2D3 by MCs2. In addition, MC-CYP27B1 activity is required for 25OHD3–induced inhibition of IgE-mediated MC-TNF and IL-6 production in vitro. Importantly, the biological significance of these findings is emphasized in our mouse model of IgE-mediated passive cutaneous anaphylaxis (PCA). By employing four mouse groups with different cutaneous MC profiles in the ears, including WT mice, MC-deficient C57BL/6-KitW-sh/W-sh mice and C57BL/6-KitW-sh/W-sh mice 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, our data provide evidence that MCs are capable of utilising 25OHD3 to generate biologically active 1α,25(OH)2D3, to efficiently repress IgE-mediated MC activation and thereby highlights the therapeutic value of altering MC function with the use of immunoregulatory agents.