Background: Keloids are disfiguring, fibrotic scar-like lesions that are challenging to treat and commonly recur after therapy. A deeper understanding of the mechanisms driving keloid formation is necessary for the development of more effective therapies. Reduced vitamin D receptor (VDR) expression has been observed in keloids, implicating vitamin D signaling in keloid pathology. Vitamin D exhibits anti-proliferative and anti-inflammatory properties, suggesting it could have therapeutic utility in keloid disorder. The current study investigated vitamin D-regulated gene expression in keloid keratinocytes and the effects of inhibiting an enzyme involved in vitamin D metabolism on the phenotype of keloid-derived keratinocytes. Methods: Normal and keloid-derived primary keratinocytes were isolated from normal skin and keloid lesions, respectively, and were cultured in the absence or presence of vitamin D. In some experiments, inhibitors of the vitamin D metabolizing enzyme CYP24A1, ketoconazole or VID400 were added in the absence or presence of vitamin D. Cellular proliferation, migration and gene expression were measured. Results: We observed significant overexpression of Conclusions: The data suggest that reduction of vitamin D inactivation with CYP24A1 inhibitors may reduce profibrotic gene expression in keloid-derived cells. Therefore, CYP24A1 inhibitors may serve as adjunctive therapies to suppress keloid-associated gene expression changes.