This study assesses the correlation between vitamin-D deficiency and autism spectrum disorder (ASD) in Jordan. We performed a case–controlled cross-sectional analysis to assess vitamin D levels in 83 children with ASD aged less than 8 years old compared to 106 healthy controls. In addition, the association between vitamin D deficiencies and gastrointestinal (GI) complains and electroencephalogram (EEG) abnormalities commonly found in children with ASD was investigated. Vitamin D levels in ASD patients were significantly lower. Also, Vitamin D levels in ASD patients had significant correlation with GI complains, but no correlation between vitamin D levels and Ca2+or EEG abnormalities was detected. These data suggest a possible role for vitamin D deficiency in the pathophysiology of ASD.