Vitamin D plays a role in central nervous system (CNS) development. Recent literature focused on vitamin D status in children and adolescents with autism spectrum disorder (ASD), but with inconsistent results. Our case-control study is aimed at evaluating serum 25-hydroxyl-vitamin D (25(OH)D) concentration in children with ASD (ASD group, n = 54) compared to children affected by other neurological and psychiatric disorders (non-ASD group, n = 36). All patients were admitted at the Complex Operative Unit of Child Neuropsychiatry, Polyclinic of Bari, Italy. 25(OH)D was quantified by chemiluminescence immunoassay and level defined as: deficiency (<20 ng/mL); insufficiency (20-30); normality (30-100); toxicity (>100). Statistical analysis was performed using SPSS20 (significance < 0.05). The ASD group showed 25(OH)D a mean level significantly lower than control (p = 0.014). Multivariable logistic regression analysis showed an association between ASD and vitamin D deficiency (p = 0.006). The nature of such association is unclear. Vitamin D deficiency may probably act as a risk factor for the development of ASD. Further studies are needed to unravel the role of vitamin D in ASD etiology and investigate its therapeutic potential.