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In many disorders requiring steroid therapy, there is substantial decrease in bone mineral density. The association between steroid use and 25-hydroxyvitamin D [25(OH)D] deficiency has not been confirmed in large population-based studies, and currently there are no specific vitamin D recommendations for steroid users.The aim of the study was to evaluate the association of serum 25(OH)D deficiency [defined as 25(OH)D <10 ng/ml] with oral steroid use.Cross-sectional analysis was performed using NHANES 2001–2006.We analyzed a nationally representative sample of U.S. children and adults.The study sample consisted of children, adolescents, and adults from NHANES 2001–2006 (n = 22,650), representative of 286 million U.S. residents, with serum 25(OH)D levels and data on other potential confounders.We measured serum 25(OH)D levels below 10 ng/ml.A total of 181 individuals (0.9% of the population) used steroids within the past 30 d. Overall, 5% of the population had 25(OH)D levels below 10 ng/ml. Among steroid users, 11% had 25(OH)D levels below 10 ng/ml, compared to 5% among steroid nonusers (P = 0.009). The odds of having 25(OH)D deficiency were 2-fold higher in those who reported steroid use compared to those without steroid use [odds ratio (OR), 2.36; 95% confidence interval (CI), 1.25, 4.45]. This association remained after multivariable adjustment (OR, 2.21; 95% CI, 1.01, 4.85) and in a multivariable model using NHANES III data (OR, 1.88; 95% CI, 1.01, 3.48).Steroid use is independently associated with 25(OH)D deficiency in this nationally representative cohort limited by cross-sectional data. It suggests the need for screening and repletion in patients on chronic steroids.