Background: Vitamin D is known to be associated with inflammatory diseases, but its relationship with allergic diseases is unclear. The study objective is to determine the association of serum vitamin D levels and markers of wheeze, asthma and atopy. Methods: Data (n = 9,463) on serum vitamin D levels and atopy were obtained from 2005-2006 National Health and Nutrition Examination Survey. Serum vitamin D level was categorized into four groups: Normal (≥30ng/ml), Insufficient (21-29ng/ml), Deficient (11-20ng/ml) and Severely Deficient (≤10ng/ml). Atopy was defined as at least 1 positive allergen-specific IgE level measured for a panel of 5 common aeroallergens- cat, dog, house dust mite, cock roach and Alternaria species. Doctor-diagnosed asthma and wheeze in the previous 12 months were assessed by means of questionnaire. Multivariable logistic regression analyses were conducted to investigate the association of serum vitamin D with wheeze, asthma and atopy adjusting for age, sex, race, smoking, outdoor physical activity, body mass index and poverty income ratio. Results: Overall, 15%, 14% and 28% of subjects had wheeze, asthma and atopy, respectively. Approximately 21% had normal serum vitamin D levels, while 35%, 28% and 5% had insufficient, deficient and severely deficient levels. Compared to subjects with normal vitamin D levels, those with insufficient, deficient and severely deficient levels had increased relative odds of wheeze and atopy with highest adjusted estimates in subjects with severe vitamin D deficiency (adjusted odds ratio [OR] 2.31, 95% Confidence Interval [CI] 1.73-3.10 for wheeze; OR 1.49, 95% CI 1.17-1.89 for atopy). Conclusion: Low serum vitamin D levels were found to be associated with wheeze and atopy. Findings contribute to ongoing efforts to understanding the role of vitamin D in atopic diseases.