Children with cancer are potentially at a high risk of plasma 25-hydroxyvitamin D (25(OH)D) inadequacy, and despite UK vitamin D supplementation guidelines their implementation remains inconsistent. Thus, we aimed to investigate 25(OH)D concentration and factors contributing to 25(OH)D inadequacy in paediatric cancer patients. A prospective cohort study of Scottish children aged <18 years diagnosed with, and treated for, cancer (patients) between August 2010 and January 2014 was performed, with control data from Scottish healthy children (controls). Clinical and nutritional data were collected at defined periods up to 24 months. 25(OH)D status was defined by the Royal College of Paediatrics and Child Health as inadequacy (<50 nmol/l: deficiency (<25 nmol/l), insufficiency (25–50 nmol/l)), sufficiency (51–75 nmol/l) and optimal (>75 nmol/l). In all, eighty-two patients (median age 3·9, interquartile ranges (IQR) 1·9–8·8; 56 % males) and thirty-five controls (median age 6·2, IQR 4·8–9·1; 49 % males) were recruited. 25(OH)D inadequacy was highly prevalent in the controls (63 %; 22/35) and in the patients (64 %; 42/65) at both baseline and during treatment (33–50 %). Non-supplemented children had the highest prevalence of 25(OH)D inadequacy at every stage with 25(OH)D median ranging from 32·0 (IQR 21·0–46·5) to 45·0 (28·0–64·5) nmol/l. Older age at baseline (R −0·46; P<0·001), overnutrition (BMI≥85th centile) at 3 months (P=0·005; relative risk=3·1) and not being supplemented at 6 months (P=0·04; relative risk=4·3) may have contributed to lower plasma 25(OH)D. Paediatric cancer patients are not at a higher risk of 25(OH)D inadequacy than healthy children at diagnosis; however, prevalence of 25(OH)D inadequacy is still high and non-supplemented children have a higher risk. Appropriate monitoring and therapeutic supplementation should be implemented.