Context: 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. Objective: 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. Design: Cross-sectional analysis was performed using NHANES 2001–2006. Setting: We analyzed a nationally representative sample of U.S. children and adults. Participants: 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. Main Outcome Measure: We measured serum 25(OH)D levels below 10 ng/ml. Results: 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). Conclusion: 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.