Background & aims: A role of vitamin D in the development of respiratory and allergic disease in children remains unclear. It may be likely that vitamin D has an effect on airway inflammation, but only few studies examined the effect in children. We aimed to examine whether serum 25-hydroxyvitamin D (25(OH) vitamin D) concentrations are associated with the fraction of exhaled nitric oxide (FeNO), airway interrupter resistance (Rint), physician diagnosed asthma ever, wheezing and eczema in a population-based cohort study in 6 year old children. Methods: Serum 25(OH) vitamin D concentration was assessed in 3815 children. 25(OH) vitamin D concentrations ≥75 nmol/L were considered as sufficient, between 50 and 75 nmol/L as insufficient, and <50 nmol/L as deficient. FeNO and Rint were measured at the research center. Data on physician diagnosed asthma, wheezing, and eczema were obtained by parent-reported questionnaires. Results: In comparison with sufficient 25(OH) vitamin D concentration, deficient concentrations were associated with elevated FeNO of ≥25 ppb (OR: 2.54; 95% CI: 1.34–4.80). In addition, deficient and insufficient 25(OH) vitamin D concentrations were associated with a lower Rint (Z-score: −1.26; 95% CI: −1.66 to −0.85) (ß: −0.75; 95% CI: −1.08 to −0.42), and increased risks of eczema (OR: 1.65; 95% CI: 1.13–2.41) (OR: 1.44; 95% CI: 1.06–1.95). Insufficient 25(OH) vitamin D concentration were associated with a decreased risk of physician diagnosed asthma ever (OR: 0.59; 95% CI: 0.38–0.94). Conclusions: Our results indicate that lower 25(OH) vitamin D levels are associated with elevated FeNO levels, but lower Rint values. Lower 25(OH) vitamin D levels are also associated with a decreased risk for asthma diagnoses but an increased risk for eczema.