Objectives: Our study sought to evaluate the association between HSV1 and serum 25-hydroxyvitamin D (25(OH) D) levels among dental students. Materials & Methods: This is a cross-sectional study with a quantitative approach conducted in Birjand, state of Khorasan Jonoobi, Iran, from July 2018 to April 2019. A peripheral blood sample (5 ml) and an anti-virus antibody along with Serum 25-hydroxyvitamin D (1,25(OH)2D3) levels were measured using an Anti-Herpes-1 IgG kit with ELISA by a pathologist. Pearson and chi-square tests were applied in the significance level of 5%. Results: The prevalence of HSV1 was 41%. The serum 25-hydroxyvitamin D3 levels mean was 23.22 ± 13.09. The prevalence of HSV1 antibody in pre-clinical and clinical levels was higher than in basic science, also, the results represented that a negative relation between vitamin D deficiency and the presence of HSV1, but it was not a significant association. Conclusion: Any association between vitamin D and herpes viruses remains inconclusive. Further studies in the general population are needed.