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Background Latent tuberculosis infection (LTBI) is a precursor of active tuberculosis diseases and an important issue in the United States and worldwide. The association between vitamin D deficiency and LTBI is poorly understood. Methods From 2011 to 2012, the National Health and Nutrition Examination Survey (NHANES) assessed LTBI (according to tuberculin skin testing and QuantiFERON ® -TB Gold In-Tube) and measured serum levels of vitamin D. We evaluated the association between LTBI and vitamin D using multivariate logistic regression models adjusted for known confounders. Results The LTBI group had a lower 25-hydroxyvitamin D [25(OH)D] level than the non-LTBI group ( p =0.0012). The adjusted risk of LTBI was significantly higher among participants with serum 25(OH)D levels <12 ng/ml (adjusted OR [aOR], 2.27; 95% CI, 1.40–3.66) and 12–19 ng/ml (aOR, 1.75; 95% CI, 1.25–2.46) compared to those with a level ≥30 ng/ml. The higher risk of LTBI among the participants with serum 25(OH)D levels <12 ng/ml and 12–19 ng/ml remained unchanged in both male and summer season subgroups. Conclusions A low serum 25(OH)D level was significantly associated with the risk of LTBI in this US cohort.