Topical application of biologically active vitamin D [1,25-dihydroxyvitamin D (1,25(OH)2D)], or low-calcemic analogues, curb skin inflammation through mechanisms that involve migratory dendritic cells (DCs) and regulatory T (TReg) cells. 1,25(OH)2D also promotes immunoregulation by mast cells, and inhibits the development of T helper type-9 (Th9) cells that secrete interleukin-9 (IL-9). Here, we investigated the ability of topical 1,25(OH)2D to suppress contact dermatitis through an IL-9-dependent process, examining mast cells and IL-9-secreting T cells. Contact dermatitis was modelled in adult BALB/c female mice by initiating a “biphasic ear swelling response” following a single application of 2,4-dinitrofluorobenzene (DNFB). Topical 1,25(OH)2D (125 ng) applied to ear pinnae prior to (but not after) DNFB sensitisation suppressed the efferent phase of the ear swelling response. This dose of 1,25(OH)2D did not cause hypercalcemia. At the peak of the efferent ear swelling response, proportions of TReg (CD3 + Foxp3+) cells and numbers of mast cells were increased in ear skin of 1,25(OH)2D-treated mice. Topical 1,25(OH)2D increased the proportion of Foxp3 + IL-9 + TReg cells and the capacity of TReg cells to secrete IL-9 ex vivo. However, the proportion of the IL-9 + cells of the total TReg cell population was small (< 1%), and the amount of IL-9 secreted by TReg cells from mice treated with IL-9 was low (< 50 pg/ml). Furthermore, injection of anti-IL-9 neutralising antibody (100 µg, intraperitoneally) prior to sensitisation did not significantly reverse the suppressive effects of 1,25(OH)2D. In conclusion, topically applied 1,25(OH)2D suppressed the efferent phase of a biphasic cutaneous ear swelling response through mechanism(s) that may be dependent on mast cells and TReg cells; however, the role of IL-9 in mediating these responses is uncertain. More studies are needed to further characterise the mechanisms by which topical 1,25(OH)2D modulates cell-mediated immune responses central to its suppressive effects upon contact dermatitis. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.