Purpose: Isotretinoin (Iso) has marked side effects. Homocysteine (Hcy) metabolizes in the liver, requiring folate and vitamin B12. Elevated blood levels of Hcy have been linked to an increased risk of premature coronary artery disease (CAD). In this study, we evaluated Hcy levels, vitamin B12, and folate in patients on Iso treatment for cystic acne (CA). Methods: Seventy-four patients with CA were included to the study group. Blood levels of Hcy, vitamin B12, and folate were assessed before and after 45 days of Iso therapy. The control group consisting of 80 individuals were tested once. Results: Hcy levels were statistically significantly increased in patients on Iso treatment. Vitamins were unaltered, while lipids and liver enzymes increased statistically significantly. Conclusion: Hcy levels are elevated in patients on Iso treatment for CA. It may be due to either the inhibition of cystathionine-beta-synthase, an enzyme required in the metabolism of Hcy, by the drug and/or the liver dysfunction. Daily supplementation with vitamin B12 and folate, which are the cofactors of the enzymatic reactions involved in Hcy metabolism, can lower plasma levels of Hcy, so it is recommended to take these vitamins in case of deficiency along with Iso to prevent premature occlusive vascular disease.