Introduction: Recurrent aphthous ulcer (RAU) is the most prevalent oral lesion. It is characterized by recurrent attacks of regular, oval or round, solitary or painful multiple ulcers. Vitamin D is a fat-soluble secosteroid pleiotropic molecule. Lower vitamin D serum level has been found to be involved in the etiology of many diseases. Objective: The aim of this study was to compare levels of serum vitamin D in patients having RAU with healthy subjects to determine if a relationship exists between vitamin D level and the severity of the disease. Subjects and Methods: 70 patients having idiopathic MiRAU (Group I) and 70 healthy control subjects with matched age and sex (Group II) were included. RAU severity was represented by number of ulcers per attack and the frequency of recurrence. 25-hydroxyvitamin D (25(OH)D) measured by enzyme-linked immunosorbent assay (ELISA). Serum vitamin D levels were compared between group I and group II and correlated with RAU severity. Results: The mean level of 25(OH)D in MiRAU group was significantly lower than the control group (p< 0.001). Severe deficiency and deficiency levels of 25(OH)D were more common in MiRAU group than in control group. Significant inverse correlation was observed between serum 25(OH)D levels and severity of RAU. Conclusions: In this study, significant association has been observed between serum vitamin D deficiency and RAU. Moreover, the strong influence of low serum vitamin D level on the severity of the disease suggesting its role in the pathogenesis of RAU.