Background: Vitamin D deficiency has been shown to be a determinant of disease severity in patients with atopic dermatitis (AD). There is a lack of information on the prevalence of vitamin D deficiency in Malaysian children with AD. The objective of this study was to determine the association of vitamin D deficiency with AD severity, to compare vitamin D deficiency between children with and without AD and to determine prevalence of vitamin D deficiency in children with AD. Methods: A case-control study to examine serum 25- hydroxyvitamin D [25(OH)D] levels in children with and without AD was done. Serum 25-hydroxyvitamin D [25(OH)D] level was measured by immunoassay. AD severity was evaluated using the SCORing Atopic Dermatitis (SCORAD) index. Results: The serum levels of 25(OH)D, measured in 135 children with AD was not statistically different from 65 children without AD [median (IQR): 25.2ng/mL (15.45) vs 25.9ng/mL (15.87), p=0.616]. However, serum vitamin D levels were significantly lower in children with severe AD compared to those with mild-to-moderate AD [median (IQR): 16.0ng/mL (19.32) vs 26.3ng/mL (15.56), p=0.021]. The odds of having vitamin D deficiency in children with severe AD was 3.82 times that of children with non-severe AD (95% confidence level: 1.13, 12.87). Conclusion: This study suggests that there is an inverse association between vitamin D level and the severity of AD in Malaysian children.