Technical Abstract: Vitamin D has immunomodulatory effects in in vitro and in animal studies. However, data from clinical studies of inflammatory diseases are scarce. The purpose of this study was to evaluate the association between serum concentrations of 25-hydroxyvitamin D [25(OH)D] and gingival inflammation. We analyzed data from 77,503 gingival units (teeth) in 6,700 never smokers aged 13 to >90 years from the third National Health Nutrition Examination Survey. Multiple logistic regression models adjusted for subject- and site-specfic covariates included age, sex, race-ethnicity, income, body mass index, diabetes, use of oral contraceptives and hormone replacement therapy among women, intake of vitamin C, missing teeth, full crown coverage, presence of calculus, frequency of dental visits, and dental examiner and survey phase. Generalized estimating equations were used to account for correlated observations within subjects. Compared with sites in subjects in the lowest 25 (OH)D quintile, sites in subjects in the highest 25 (OH)D quintile were 20% (95% CI: 8%, 31%) less likely to bleed on gingival probing (P for trend <0.001). The association appeared to be linear over the entire 25(OH)D range, was consistent across racial and ethnic groups, and was similar among men and women as well as among users and nonusers of vitamin and mineral supplements. In conclusion, Vitamin D may reduce susceptibility to gingival inflammation through its anti-inflammatory effects. Gingivitis may be a useful clinical model to evaluate the anti-inflammatory effects of vitamin D.