BACKGROUND Vitamin D plays an important role in etiology of Autism Spectrum Disorders (ASDs). We aimed to evaluate the serum 25 - hydroxyl vitamin D level among children with ASDs in Ahvaz city, Iran. METHODS It was a cross-sectional study which had conducted on 62 subjects in two groups: a case group (n = 31) consisted of ASD children who study in especial schools; and a control group (n = 31) of healthy children who were selected by simple random sampling from regular schools in Ahvaz city, Iran during 2016. Maching between two groups has done regarding Socioeconomic status, type and amount of food intake, place of living and age. The levels of serum 25 - hydroxyl vitamin D were assessed in early morning means fasted state and also measured using ELISA method. Data were analyzed using Statistical Package for Social Sciences (SPSS) version 20. The significant level was considered < 0.05. RESULTS In ASD children, the average serum 25-hydroxyvitamine D level was 9.03 ± 4.14 ng/mg. In ASD group, 96.8% (30 subjects) had vitamin D deficiency. In healthy children group, average serum 25-hydroxyvitamine D level was 15.25 ± 7.89 ng/mg. Average serum 25-hydroxyvitamine D level in intervention group was significantly lower than the control group (P > 0.001). Although the parents of patients in control group reported longer exposure to sun (27.42 m per day against 33.06 m per day), no significant difference was observed between these groups in terms of exposure to sun (P < 0.05). CONCLUSIONS A significant difference was observed between serum 25-hydroxyvitamine D levels between the healthy and ASD children. It is recommended to use vitamin D supplement in children with ASDs under medical care.