Epidemiological evidence has shown that pediatric food allergy is more prevalent in regions further from the equator, suggesting that vitamin D insufficiency may play a role in this disease. To investigate the role of vitamin D status in infantile food allergy. A population sample of 5276 one-year-old infants underwent skin prick testing to peanut, egg, sesame, and cow's milk or shrimp. All those with a detectable wheal and a random sample of participants with negative skin prick test results attended a hospital-based food challenge clinic. Blood samples were available for 577 infants (344 with challenge-proven food allergy, 74 sensitized but tolerant to food challenge, 159 negative on skin prick test and food challenge). Serum 25-hydroxyvitamin D levels were measured by using liquid chromatography tandem mass spectrometry. Associations between serum 25-hydroxyvitamin D and food allergy were examined by using multiple logistic regression, adjusting for potential risk and confounding factors. Infants of Australian-born parents, but not of parents born overseas, with vitamin D insufficiency (≤50 nmol/L) were more likely to be peanut (adjusted odds ratio [aOR], 11.51; 95% CI, 2.01-65.79; P=.006) and/or egg (aOR, 3.79; 95% CI, 1.19-12.08; P=.025) allergic than were those with adequate vitamin D levels independent of eczema status. Among those with Australian-born parents, infants with vitamin D insufficiency were more likely to have multiple food allergies (≥2) rather than a single food allergy (aOR, 10.48; 95% CI, 1.60-68.61 vs aOR, 1.82; 95% CI, 0.38-8.77, respectively). These results provide the first direct evidence that vitamin D sufficiency may be an important protective factor for food allergy in the first year of life.