Abstract Background Patients with erythropoietic protoporphyria experience lifelong painful photosensitivity resulting in a lack of sunlight exposure. Previous studies have shown that 47–63% of patients with EPP suffer from vitamin D deficiency and a high prevalence of osteoporosis. An effective treatment for EPP has been available since 2016: the α-melanocyte stimulating hormone analogue afamelanotide. So far, studies on vitamin D levels in EPP have only investigated patients who have not been treated with afamelanotide. Objectives To investigate the effects of afamelanotide treatment on vitamin D levels in EPP. Methods A multicentre observational cohort study in adults with EPP from the Erasmus Medical Centre, the Netherlands, and the University Hospital Düsseldorf, Germany, was carried out. Routinely collected vitamin D levels between 2005 and 2021 were used for analysis. Patient exposure to cholecalciferol or afamelanotide was categorized into four treatment groups: untreated, cholecalciferol, afamelanotide and combined treatment. A linear mixed model for longitudinal data was applied to measure the effect of the treatment groups compared with the untreated groups on vitamin D levels. Results A total of 230 patients and 1774 vitamin D measurements were included. The prevalence of vitamin D deficiency and severe deficiency remained high despite afamelanotide treatment (< 50 nmol L–1 in 71.8% of patients and < 30 nmol L–1 in 48.1%, respectively). Afamelanotide treatment alone did not lead to a significant average increase in vitamin D levels [β = 0.5, 95% confidence interval (CI) –3.2 to 4.2]. In contrast, cholecalciferol and combined therapy with afamelanotide led to a significant increase in vitamin D levels [β = 11.6 (95% CI 7.2–15.9) and β = 15.2 (95% CI 12.3–18.1), respectively]. Conclusions Cholecalciferol remains essential for the treatment of vitamin D deficiency in EPP, irrespective of new treatment options like afamelanotide. Afamelanotide treatment did not affect vitamin D levels. We suggest that future guidelines include continuous monitoring of vitamin D and a prescription for cholecalciferol in all patients with EPP, including those treated with afamelanotide.