Background: Determinants of vitamin D status are of interest when studying the epidemiology of disease in population groups because vitamin D is now recognized to decrease the risk of diseases such as osteoporosis, cancer, and cardiovascular disease. Understanding modifiable determinants of vitamin D status are important for managing vitamin D deficiency at the individual level and for addressing this issue at population level. Objective: The objective of this study was to evaluate the associations between serum vitamin D status (deficiency and insufficiency) and distinct demographic, dietary, and lifestyle characteristics of adults in the United States using a large, nationally representative sample survey, the National Health and Nutrition Examination Survey (NHANES) 2005-2006. Methods: The study sample consisted of 2340 adults aged 20-59 who had serum 25(OH)D measured and who had completed various questionnaires concerning dietary intake of vitamin D and other lifestyle factors. Multivariate logistic regression was used to estimate the odds ratio (OR) of vitamin D deficiency, insufficiency, and sufficiency in adults based on distinct demographic, dietary, and lifestyle characteristics. Statistical significance was set at α < 0.05. Results: The prevalence of vitamin D deficiency was higher in obese adults than in underweight to normal weight adults (50.9% ± 4.57 vs. 29.3% ± 3.57), higher in adults who reported no sunburns than in adults who reported ≥ 3 sunburns (49.9% ± 3.82 vs. 18.0% ± 3.07), and higher in adults who use sun protective measures regularly than in adults who do not (48.4% ± 3.93 vs. 27.0% ± 3.75). The prevalence of vitamin D deficiency increased as dietary intake of vitamin D decreased. Non-Hispanic black adults were significantly more likely to be vitamin D deficient (OR = 45.27, 95% CI = 17.27-118.64) and insufficient (OR = 9.37, 95% CI = 3.43-25.61) than non-Hispanic white adults. Significant positive associations were found between vitamin D deficiency and several characteristics, namely obesity (OR = 7.43, 95% CI = 4.33-12.77), physical inactivity (OR = 1.63, 95% CI = 1.03-2.58) poor dietary vitamin D intake (OR = 2.34, 95% CI = 1.44-3.81), non-supplement use or supplement use with a low amount of vitamin D (OR = 1.75, 95% CI = 1.05-2.89), and activities that decrease exposure to sunlight (from OR = 2.97, 95% CI = 2.14-4.13 to OR = 5.30, 95% CI = 3.17-8.85). Conclusion: The results of this nationally representative study demonstrate that obesity, physical inactivity, poor dietary intake of vitamin D, and low sunlight exposure increases the risk for vitamin D deficiency in U.S adults. Future studies are needed to investigate whether vitamin D supplementation, sunlight exposure, and vitamin D-fortified foods are efficient in correcting vitamin D deficiency and insufficiency among these groups.