Purpose: The doubling in the prevalence of myopia worldwide over the last few decades represents a significant public health concern and argues for significant environmental influences. While outdoor activity appears to play a protective role in the development of myopia, the underlying mechanism, including the role of Vitamin D, remains unresolved. The large National Health and Nutrition Examination Survey (NHANES) open-access dataset offers unique opportunity to address the latter issue. Methods: The study made use of data from NHANES (2003-2008), including participants aged 12-40 years with complete vision examination and personal characteristic data. In addition to demographic information, serum Vitamin D levels, refractive error (non-cycloplegic Nidek) and limited questionnaire data on outdoor activity and device use (computer or TV) were extracted. Associations between serum Vitamin D level and presence and severity of myopia (defined as -0.75 D or worse) were evaluated using multivariate regression. Results: Data from 9,349 ethnically diverse participants were used in analyses, with approximately 50% having myopia. The percentage of participants with low Vitamin D (defined by median split, 54.4 nmol/L) varied significantly (high Vitamin D levels to be mildly protective OR=0.77). Conclusion: Low Vitamin D does not appear to be a risk factor for the severity of myopia, but may be a risk factor for the development of myopia, especially at younger ages. Longitudinal pediatric studies, beginning prior to the onset of myopia, are needed to better understand the apparent protective effect of outdoor activity and to identify modifiable risk factors for myopia development.