Vitamin D is important for normal skeletal homeostasis, especially in growing children. There are no previous genome-wide association (GWA) studies exploring genetic factors that influence vitamin D metabolism in early childhood. We performed a GWA study on serum 25-hydroxyvitamin D (25(OH)D) and response to supplementation in 761 healthy term-born Finnish 24-month-old children, who participated in a randomized clinical trial comparing effects of 10 μg and 30 μg of daily vitamin D supplementation from age 2 weeks to 24 months. Using the Illumina Infinium Global Screening Array, which has been optimized for imputation, a total of 686085 markers were genotyped across the genome. Serum 25(OH)D was measured at the end of the intervention at 24 months of age. Skeletal parameters reflecting bone strength were determined at the distal tibia at 24 months using peripheral quantitative computed tomography (pQCT) (data available for 648 children). For 25(OH)D, two strong GWA signals were identified, localizing to GC (Vitamin D binding protein) and CYP2R1 (Vitamin D 25-hydroxylase) genes. The GWA locus comprising the GC gene also associated with response to supplementation. Further evidence for the importance of these two genes was obtained by comparing association signals to gene expression data from the Genotype-Tissue Expression project and performing colocalization analyses. Through the identification of haplotypes associated with low or high 25(OH)D concentrations we used a Mendelian randomization approach to show that haplotypes associating with low 25(OH)D were also associated with low pQCT parameters in the 24-month-old children. In this first GWA study on 25(OH)D in this age group we show that already at the age of 24 months genetic variation influences 25(OH)D concentrations and determines response to supplementation, with genome-wide significant associations with GC and CYP2R1. Also, the dual association between haplotypes, 25(OH)D and pQCT parameters gives support for vertical pleiotropy mediated by 25(OH)D.