Mast cells (MCs) are typically regarded as pro-inflammatory effecter cells in immunoglobulin (Ig)E-dependent allergic disorders. However, MCs can have anti-inflammatory properties in response to application of 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃), the biologically active form of vitamin D3 (VitD₃). In the presence of 1α,25(OH)₂D₃, IgE + specific Ag (sAg)-stimulation in bone marrow-derived MCs (BMCMCs) resulted in a suppressed production of pro-inflammatory cytokines, TNF and IL-6, in a vitamin D receptor (VDR)-dependent manner. Moreover, the presence of 1α,25(OH)₂D₃ can induce the production of anti-inflammatory cytokine IL10 from mouse BMCMCs. In addition, we have shown that mouse BMCMCs have the ability to produce 1α,25(OH)₂D₃ from the inactive VitD₃ metabolite, 25-hydroxyvitamin D3 (25OHD₃), due to expression and activity of the hydroxylase enzyme CYP27B1. 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-sh mice and C57BL/6-Kit^W-sh/W-sh mice engrafted with either WT or VDR^-/- BMCMCs, we found that topical application of 1α,25(OH)₂D₃ or 25OHD₃ significantly reduced the magnitude of PCA-associated ear swelling. Taken together, these effects required the presence of VDR-expressing dermal MCs. Our findings provide evidence that 1α,25(OH)₂D₃, as well as 25OHD₃, can suppress IgE + sAg-mediated MC activation in a VDR-dependent manner both in vitro and in vivo, and suggest that 1α,25(OH)₂D₃ may have therapeutic potential for treatment of MC-dependent IgE-associated allergic disorders.