This study aimed to investigate the association between 25-hydroxyvitamin D (25(OH)D) level and low back pain (LBP) among adolescents while adjusting for potential confounders pertinent to this age group including the weight of school bags, BMI and physical activity. A cross-sectional study was conducted on 760 randomly selected adolescents in middle schools. Data on LBP and the risk factors for LBP were collected from parents by a self-administered questionnaire and from adolescents by face-to-face interview. Blood samples were tested in an accredited laboratory; and 25(OH)D was measured using liquid chromatography-tandem MS. The lifetime prevalence and the 6-month prevalence of LBP were 32·28 (95 % CI 28·97, 35·73) % and 21·26 (95 % CI 18·40, 24·33) %, respectively. There was no difference in the geometric mean of 25(OH)D between those with and without LBP in the past 6 months (28·50 nmol/l and 30·82 nmol/l, respectively; P = 0·122). There was no association between 25(OH)D and LBP in the univariable or multivariable analysis whether 25(OH)D fitted as a continuous or as a categorical variable. We found no association between vitamin D level and LBP in adolescents in an area with high prevalence of vitamin D deficiency. Although it is important to have sufficient vitamin D levels during adolescence for several other health benefits, we concluded that vitamin D is not a major determinant for LBP among adolescents in our setting.