Abstract Although vitamin D status may be a modifiable risk factor for various respiratory ailments, limited data exists regarding its role in sinonasal infections. Our goal was to investigate the association of 25-hydroxyvitamin D (25OHD) levels with acute rhinosinusitis (ARS) in a large, nationally representative sample of non-institutionalized individuals from the United States. In this cross-sectional study of individuals ≥17 years from the National Health and Nutrition Examination Survey 2001–2006, we used multivariable regression analysis to investigate the association of 25OHD levels with ARS, while adjusting for season, demographics (age, sex, race, and poverty-to-income ratio), and clinical data (smoking, asthma, chronic obstructive pulmonary disease, diabetes mellitus, and neutropenia). A total of 3921 individuals were included in our analyses. Median 25OHD level was 22 (interquartile range 16–28) ng/mL. Overall, 15.8% (95% confidence interval [CI] 14.4–17.7) of participants reported ARS within the 24 hours leading up to their survey participation. After adjusting for season, demographics, and clinical data, 25OHD levels were associated with ARS (odds ratio 0.88, 95% CI 0.78–0.99 per 10 ng/mL). When vitamin D status was dichotomized, 25OHD levels <20 ng/mL were associated with 33% higher odds of ARS (odds ratio 1.33, 95% CI 1.03–1.72) compared with levels ≥20 ng/mL. Our analyses suggest that 25OHD levels are inversely associated with ARS. Randomized, controlled trials are warranted to determine the effect of optimizing vitamin D status on the risk of sinonasal infections