We report a case of Hailey-Hailey disease (HHD) in which 1α,24-dihydroxyvitamin D3 (tacalcitol) was effective both clinically (in vivo) and in explant cultures (in vitro) of a skin lesion. The patient was a 65-year-old man with HHD lesions in the axillary and inguinal areas bilaterally. We applied ointment containing 1α,24-dihydroxyvitamin D3 (tacalcitol), an analogue of active vitamin D3, to the lesions and assessed its clinical effectiveness. The HHD lesions in both groins disappeared after treatment with the 1α,24-dihydroxyvitamin D3 ointment, and the remission has continued to the present. A punch biopsy specimen of the lesion that had remitted showed no acantholysis. In addition, dissociation of migrating keratinocytes was observed when biopsy specimens of the HHD skin lesion were cultured in medium without 1α,24-dihydroxyvitamin D3, but inhibition of keratinocyte dissociation was observed in medium containing it. These results suggest the effectiveness of 1α,24-dihydroxyvitamin D3 against HHD both in vivo and in vitro.