Sleep problems are among the most prevalent comorbidities experienced by children with Autism Spectrum Disorder (ASD). There is a clinical and physiological basis for a link between 25(OH) D levels and sleep disorders. In this study we aimed to investigate the frequency of sleep disorders in ASD patients and its association with 25(OH) D levels, and whether or not these frequencies changed after 25(OH) D treatment. This prospective study included 60 consecutive patients diagnosed with ASD and matched healthy controls between the ages of 4 and 10. Patients then underwent 25(OH) D replacement therapy according to their deficiency levels. Pre- and post-therapy values were compared. Sleep disturbance was detected in 78.3% of ASD patients (n = 60) and 33.3% of the control group (n = 60). When we compared the pretreatment scores of sleep disturbance between ASD and control groups (n = 60), there were significant differences in bedtime resistance, sleep anxiety, parasomnias, daytime sleepiness, sleep duration, sleep-onset delay, night wakings subscales, and total scale score (p < 0.05); however there were no significant differences with respect to the sleep-disordered breathing subscales (p > 0.05). In ASD patients, there was a significant negative correlation between serum 25(OH) D levels and the night wakings subscale (r = -0.301, p = 0.019). In control patients, there was a significant negative correlation between serum 25(OH) D levels and daytime sleepiness subscales (r = -0.269, p = 0.038). The results indicate that it may be suitable to use 25(OH) D replacement therapy in ASD patients and healthy individuals with sleep disturbances.