Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of the BCR‑ABL1 fusion gene, a constitutively active, oncogenic tyrosine kinase that is responsible for the clinical features of CML. Tyrosine kinase inhibitors, such as imatinib, have markedly altered the treatment of CML. However, tyrosine kinase inhibitors are associated with side effects on bone metabolism, in adult and pediatric patients. Vitamin D3 is involved in the complex cycle of bone remodeling, therefore the present study aimed to investigate the influence of imatinib on vitamin D3 metabolism in the HaCaT human keratinocyte cell line, using commercially available enzyme assays. Imatinib was shown to significantly reduce the production of calcidiol and calcitriol. Based on interaction studies of imatinib with the cytochrome P450 (CYP450) inhibitors VID400 and ketoconazole, it is proposed that imatinib may interfere with the vitamin D3 cascade due to its metabolism by CYP27B1, which is involved in vitamin D3 metabolism.