Objective: To investigate the characteristics of 25(OH)D level in children with ASD and its correlation with clinical features. Methods: A total of 196 children with ASD who received outpatient and inpatient rehabilitation training from January 2017 to January 2019 were included in ASD group, and 178 healthy children who visited the hospital during the same period were included in healthy control group. Differences in 25(OH)D levels and general data between study group and healthy control group were compared. In addition, ASD group was divided into 25(OH)D normal group and abnormal group in accordance with 25(OH)D level (≥30 ng/mL). Differences in general data, total score of CARS scale and factor scores were compared between two groups. Finally, the correlation between serum 25(OH)D level and CARS total score and factor scores of children with ASD was evaluated. Results: 25(OH)D level in ASD group was significantly lower than that in healthy control group (P<0.01). The incidence of sleep disorder, dietary bias, vomiting, constipation and diarrhea in children with ASD was statistically significant compared with that of healthy children (P<0.01); there were statistically significant differences in breastfeeding, sleep disorder, dietary bias and diarrhea between 25(OH)D normal group and abnormal group (χ2=4.97，8.69,6.67,3.98,P<0.05); there were statistically significant differences in 10 aspects including CARS total score, interpersonal relationship, imitation, emotional response, physical use ability, relationship with inanimate objects, adaptation to environmental changes, visual response, auditory response and general impression (P<0.05); there was a significant negative correlation between serum 25(OH)D level and CARS total score in children with ASD (r=-0.32, P<0.01). Conclusion Breastfeeding could reduce the risk of 25(OH)D abnormalities in children with ASD. 25(OH)D reduction would cause sleep disorder, dietary bias and gastrointestinal problems, while dietary bias and gastrointestinal problems would affect 25(OH)D uptake and absorption. 25(OH)D might be related to the occurrence of ASD in children. Serum 25(OH)D level could be used as a reference index for the severity of ASD in children.