Background: Epidemiological studies suggest a link between vitamin D deficiency in early life and development of asthma in later life. Aim: The aim of this study was to measure serum vitamin D levels in asthmatic children and to compare these to healthy non-asthmatic controls. Methods: Asthmatic (n = 483) and healthy control (n = 483) children were recruited from the Pediatric Allergy-Immunology Clinics of Hamad General Hospital and the Primary Health Care Clinics in Qatar from October 2009 to July 2010. All children were below 16 years of age and asthma was diagnosed by a physician. Parents of all children completed extensive questionnaires documenting demographics, child’s feeding practice and vitamin D intake. Serum vitamin D (25-hydroxyvitamin D), calcium, phosphorus, alkaline phosphatase, magnesium, creatinine and parathyroid hormone assays were performed. Subjects with serum containing less than 20 ng/ml vitamin D were deemed deficient. Results: Asthmatic children had significantly reduced serum vitamin D levels compared to non-asthmatic children (p < 0.001); 68.1% of all asthmatics were vitamin D deficient. Asthmatic children had significantly higher degrees of moderate (41.8 vs. 25.1%) and severe (26.3 vs. 11.0%) vitamin D deficiency compared to healthy controls (p < 0.001). Positive familial history of vitamin D deficiency (35.6%, p = 0.005) and asthma (36.4%, p = 0.009) were significantly higher in asthmatic children. Along with vitamin D deficiency, asthmatics also had reduced phosphorus (p < 0.001) and magnesium (p = 0.001) levels but elevated serum alkaline phosphatase (p < 0.001) and IgE (p < 0.001). The majority of asthmatic children had less exposure to sunlight (66.7%, p = 0.006) and less physical activity (71.3%, p < 0.001). Vitamin D deficiency was the strongest predictor of asthma in this population (OR 4.82; 95% CI 2.41–8.63, p < 0.001). Conclusion: The present study revealed that the majority of asthmatic children had vitamin D deficiency compared to control children. Vitamin D deficiency was the major predictor of asthma in Qatari children.