Isotretinoin is an effective therapy for severe nodulocystic acne. Several experimental studies suggest that it may have an effect on vitamin D physiology. In the present study, the authors aimed to investigate the effect of isotretinoin treatment on the metabolism of vitamin D in acne patients. A prospective analysis of 50 consecutive acne patients who were treated with isotretinoin for 3 months was done. Before and after 3 months of treatment, 25 hydroxy vitamin D, 1,25 dihydroxy vitamin D, and bone alkaline phosphatase, calcium, phosphate, and parathormone levels were measured. The 25 hydroxy vitamin D and serum calcium levels decreased significantly (p < 0.0001, p < 0.05, respectively), whereas 1,25 dihydroxy vitamin D, parathormone, and bone alkaline phosphatase levels increased significantly after 3 months of isotretinoin treatment (p < 0.005, p < 0.005, p < 0.0001, respectively). Aspartate aminotransferase, total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels also increased significantly after isotretinoin treatment. This prospective clinical study showed that isotretinoin has an effect on vitamin D metabolism. Further clinical studies with longer periods of follow-up are needed to understand the effect of isotretinoin on vitamin D and bone metabolism.