Background and PurposeThe promotion of hair regeneration and growth heavily depends on the activation of Wnt/β‐catenin signalling in the hair follicle, including dermal papilla (DP). KY19382, one of the newly synthesized analogues of indirubin‐3′‐monoxime (I3O), was identified as a Wnt/β‐catenin signalling activator via inhibition of the interaction between CXXC‐type zinc finger protein 5 (CXXC5) and dishevelled (Dvl). Given the close relationship between the Wnt/β‐catenin signalling and hair regeneration, we investigated the effect of KY19382 on hair regrowth and hair follicle neogenesis.Experimental ApproachIn vitro hair induction effects of KY19382 were performed in human DP cells. The hair elongation effects of KY19382 were confirmed through the human hair follicle and vibrissa culture system. In vivo hair regeneration abilities of KY19382 were identified in three models: hair regrowth, wound‐induced hair follicle neogenesis (WIHN) and hair patch assays using C57BL/6 mice. The hair regeneration abilities were analysed by immunoblotting, alkaline phosphatase (ALP) and immunohistochemical staining.Key ResultsKY19382 activated Wnt/β‐catenin signalling and elevated expression of ALP and the proliferation marker PCNA in DP cells. KY19382 also increased hair length in ex vivo‐cultured mouse vibrissa and human hair follicles and induced hair regrowth in mice. Moreover, KY19382 significantly promoted the generation of de novo hair follicles as shown by WIHN and hair patch assays.Conclusion and ImplicationsThese results indicate that KY19382 is a potential therapeutic drug that exhibits effective hair regeneration ability via activation of the Wnt/β‐catenin signalling for alopecia treatments.