Introduction Nitric oxide (NOx) is an important biomolecule which interacts with other molecules including 25(OH)D to mediate various metabolic pathways. Interactions and associations of NOx with 25(OH)D have been well studied both in vitro and in vivo, yet associations in apparently healthy adolescents have never been studied. Methods A total of 740 (245 boys and 495 girls) apparently healthy Saudi adolescents aged 10-17 years were included in this cross-sectional study, to determine the associations of NOx with 25(OH)D and other biomarkers in Saudi adolescents. Serum NOx, 25(OH)D, and other biochemical and anthropometric parameters were measured following standard protocols and manufacturers' guidelines. Results NOx level was significantly higher in boys than girls (p<0.001). In all subjects, NOx showed a significant inverse correlation with 25(OH)D. After stratification according to sex however this significant association was observed only in boys and not in girls. NOx was also significantly associated with BMI, serum triglycerides, and systolic blood pressure in all subjects. Conclusion The significantly inverse association of NOx and 25(OH)D among apparently healthy adolescents is influenced by sex and further strengthens the extraskeletal role of 25(OH)D in maintaining endothelial homeostasis in this age group, particularly in boys. Whether vitamin D correction can influence NOx production over time among adolescents remains to be proven.