Objectives: Autism spectrum disorder (ASD) is a developmental disorder characterized by pervasive deficits in social interaction, impairment in verbal and nonverbal communication, and stereotyped patterns of interests and activities. Vitamin-D deficiency was previously reported in autistic children. However, the data on the relationship between vitamin D deficiency and the severity of autism are limited. Methods We performed a case-controlled cross-sectional analysis conducted on 122 ASD children, to assess their vitamin D status compared to controls and the relationship between vitamin D deficiency and the severity of autism. We also conducted an open trial of vitamin D supplementation in ASD children. Results Fifty-seven percent of the patients in the present study had vitamin D deficiency, and 30% had vitamin D insufficiency. The mean 25-OHD levels in patients with severe autism were significantly lower than those in patients with mild/moderate autism. Serum 25-OHD levels had significant negative correlations with Childhood Autism Rating Scale (CARS) scores. Of the ASD group, 106 patients with low-serum 25-OHD levels (<30 ng/ml) participated in the open label trial. They received vitamin D3 (300 IU/kg/day not to exceed 5000 IU/day) for 3 months. Eighty-three subjects completed 3 months of daily vitamin D treatment. Collectively, 80.72% (67/83) of subjects who received vitamin D3 treatment had significantly improved outcome, which was mainly in the sections of the CARS and aberrant behavior checklist subscales that measure behavior, stereotypy, eye contact, and attention span. Conclusion Vitamin D is inexpensive, readily available and safe. It may have beneficial effects in ASD subjects, especially when the final serum level is more than 40 ng/ml. Trial registration number UMIN-CTR Study Design: trial Number: R000016846.