Purpose Anorexia nervosa (AN) is an eating disorder characterized by low fat mass complicated by osteoporosis. The role of circulating vitamin D in the development of bone loss in AN is unclear. Fat mass is known to be inversely associated with vitamin D levels measured as serum levels of total, protein-bound 25-hydroxyvitamin D, but the importance of directly measured, free levels of 25(OH)D has not been determined in AN. The aim of this study was to investigate vitamin D status, as assessed by serum concentrations of total and free serum 25(OH)D in patients with AN and healthy controls. Methods In female AN patients (n = 20), and healthy female controls (n = 78), total 25(OH)D was measured by LC-MS/MS, and free 25(OH)D with ELISA. In patients with AN bone mineral density (BMD) was determined with DEXA. Results There were no differences between patients and controls in total or free S-25(OH)D levels (80 +/- 31 vs 72 +/- 18 nmol/L, and 6.5 +/- 2.5 vs 5.6 +/- 1.8 pg/ml, respectively), and no association to BMD was found. In the entire group of patients and controls, both vitamin D parameters correlated with BMI, leptin, and PTH. Conclusions The current study did not demonstrate a vitamin D deficiency in patients with AN and our data does not support vitamin D deficiency as a contributing factor to bone loss in AN. Instead, we observed a trend toward higher vitamin D levels in AN subjects compared to controls. Measurement of free vitamin D levels did not contribute to additional information.