Background Children and adolescents with leukemia are potentially at high risk of vitamin D inadequacy, which may have clinical relevance for skeletal morbidity, infections, and cancer outcome. This study aimed to evaluate vitamin D status at the time of diagnosis to investigate its predictors and association with overall survival in children with leukemia. Procedure We included all 295 children and adolescents diagnosed with leukemia at our institution between 1990 and 2016 who had available serum sample from the time of diagnosis. We analyzed serum 25-hydroxyvitamin D and parathyroid hormone levels and correlated them with clinical data. Results The 25-hydroxyvitamin D level was deficient (amp;lt; 25 nmol/L), insufficient (25-50 nmol/L), sufficient (50-75 nmol/L), and optimal (amp;gt; 75 nmol/L) in 6.4%, 26.8%, 39.7%, and 27.1% of the children, respectively. Older age and a more recent time of sampling (calendar year) predicted lower 25-hydroxyvitamin D level. In preschool children (age amp;lt;= 6 years), lower 25-hydroxyvitamin D level was also associated with acute myeloid leukemia, and a 25-hydroxyvitamin D level amp;lt; 50 nmol/L was associated with inferior overall survival. In school-aged children (age amp;gt; 6 years), the 25-hydroxyvitamin D level showed significant seasonal variation. Conclusion It remains unclear whether vitamin D supplementation in pediatric leukemia patients will improve outcome.