Background: Vernal keratoconjunctivitis (VKC) is a chronic and often severe bilateral conjunctivitis. VKC etiology still remains unclear although endocrine, genetic, neurogenic and environmental factors have been implicated. Vitamin D is a fat-soluble prohormone whose main function is the regulation of calcium and phosphate metabolism. The aim of this study was to evaluate serum vitamin D in children affected by VKC compared to the healthy children and investigate the relationship between its levels and disease severity. Methods: A total of 110 children, 47 affected by VKC, aged between 5 and 12 years were enrolled at the Department of Pediatrics, Division of Allergy and Immunology, "Sapienza" University of Rome. Used as controls were 63 healthy children with negative skin prick test (SPT), without allergic, ocular and systemic disease. Serum samples were obtained in April from all the children included in the study. Vitamin D dosage was repeated in October in 20 patients after therapy and in 20 controls. A conjunctival scraping was performed in all children affected by VKC. Results: Children affected by VKC had lower vitamin D levels compared to healthy controls and we found an increase in vitamin D levels after therapy with cyclosporine eye drops 1% although this increase was lower than that of healthy controls. Moreover we found significant correlations between vitamin D level and the severity of the disease. Conclusions: The study shows that children affected by VKC have lower vitamin D levels when compared to healthy controls and highlights a significant correlation between its levels and disease severity.