Purpose: We aimed to examine the association between 25-hydroxyvitamin D [25(OH)-D] and anemia (or iron deficiency) in a nationally representative sample of Korean children and adolescents. Methods: Cross-sectional data on 2,526 children and adolescents aged 10–20 years from the Korea National Health and Nutrition Examination Survey-V (2010–2012) were used. Anemia was defined according to specifications of the World Health Organization. Iron deficiency was defined as serum ferritin level of 12 ng/mL and transferrin saturation 16%. Results: The prevalence of anemia in all subjects was 3.8% (male vs. female, 1.1% vs. 6.8%; i＞기울림꼴/i＞Pi＞기울림꼴/i＞0.001). Female subjects had a higher prevalence of vitamin D deficiency (50.9% vs. 35.7%). The median (interquartile range) 25(OH)-D level in all subjects was 15.9 ng/mL (12.7-19.4 ng/mL). In logistic regression, risk factors for anemia were female sex, age, post-menarche, low household income, and vitamin D deficiency. The lowest 25(OH)-D quartile levels (12.7 ng/mL) were associated with increased risk for anemia and iron deficiency (adjusted odds ratio [95% confidence interval], 1.80 [1.10–2.95], i＞기울림꼴/i＞Pi＞기울림꼴/i＞=0.019, and 1.63 [1.03–2.59], i＞기울림꼴/i＞Pi＞기울림꼴/i＞=0.038). Second quartile levels (15.9 ng/mL) were also associated with increased risk. In linear regression, small but significant increases in hemoglobin were noted in the upper quartiles of 25(OH)-D compared with the lowest quartile (12.7 ng/mL) only in female subjects. Conclusions: Deficiency in 25(OH)-D was associated with increased risk of anemia and iron deficiency in healthy female children and adolescents. Further studies are needed to determine whether 25(OH)-D supplementation could correct anemia and iron deficiency.