Vitamin D affects the absorption of folate in vitro, and perhaps of vitamin B12 (B12). However, epidemiological studies on the association of vitamin D with folate and B12 are inconclusive. We hypothesized a positive association of plasma 25-hydroxyvitamin D [25(OH)D] with folate and B12 levels in adolescents. This hypothesis was tested in a cross-sectional study of healthy adolescents (11-16 years old; n = 1416), selected from public middle schools from across Kuwait, using stratified multistage cluster random sampling. Plasma 25(OH)D was measured by LC-MS/MS. Serum B12 and total folate in hemolyzed whole blood were analyzed with commercial kits; RBC and plasma folate were calculated from total folate. Data on potential confounders were collected from the parents and adolescents. In a univariable model, 25(OH)D as a continuous variable was positively associated with each of total, RBC, and plasma folate (P < .001). After adjusting for potential confounders, this association remained significant with total folate (β = 2.0, P < .001) and red blood cell folate (β = 1.8, P < .001), but not with plasma folate (β = 0.2, P = .34). A similar pattern of association was evident when 25(OH)D was fitted as categorical variable. Correlation between B12 and 25(OH)D was weak but significant (ρ = 0.1, P < .001). 25(OH)D was positively associated with B12 in both univariable and multivariable models (P < .001) when fitted as a categorical variable only. Simultaneous quantile regression confirmed these results. We conclude that plasma 25(OH)D is positively associated with folate and B12 levels in adolescents. Properly designed large-scale randomized controlled trials are warranted to investigate the causal role of vitamin D in folate and B12 absorption.