Evidence indicates low 25-hydroxyvitamin D [(25(OH)D] concentrations in European adolescents. Identification of potential determinants is therefore essential to guide public health initiatives aiming at optimizing vitamin D status across Europe. The aim of the study was to identify potential influencing factors of 25(OH)D concentrations in European adolescents aged 12.5 to 17.5 y, participating in the multi-centre cross-sectional Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) study. A subset of 1,006 participants (46.8% males) was drawn from the main study. Measures of body composition, biochemical markers, socioeconomic status, dietary intake, physical activity, fitness, sleep time and vitamin D genetic polymorphism (rs1544410) were assessed. Stepwise multivariate linear regression analysis was conducted stratified by gender. In males, linear regression of 25(OH)D, suggested that (1) winter season (β=-0.364; p<0.01), (2) higher latitudes (β=-0.246; p<0.01), (3) BMI z-score (β=-0.198; p<0.05) and (4) retinol concentration (β=0.171; p<0.05) independently influenced 25(OH)D concentrations. In females, (1) winter season (β=-0.370; p<0.01), (2) sleep time (β=-0.231; p<0.01), (3) supplement intake (β=0.221; p<0.05), (4) flexibility (β=0.184; p<0.05), (5) body fat % (β=0.201; p<0.05) (6), BMI z-score (β=-0.272; p<0.05), (7) higher latitudes (β=-0.219; p<0.01) and (8) handgrip strength (β=0.206; p<0.05) independently influenced 25(OH)D concentrations. Season, latitude, fitness, adiposity, sleep time and micronutrient supplementation were highly related to 25(OH)D concentrations found in European adolescents.