Background Iron deficiency anemia, pregnancy, and end-stage renal disease (ESRD) are common causes of secondary restless legs syndrome (RLS). Serum ferritin is considered the most specific test associated with the total amount of body iron stores. However, due to the increase of serum ferritin secondary to inflammation in chronic hemodialysis (HD) patients, serum ferritin test results do not fully reflect decreased iron stores in these patients. The present study evaluates the serum hepcidin levels, as the main regulator of iron metabolism, and its relationship with RLS in chronic HD patients. Methods The present cross-sectional study involved 72 patients (36 with and 36 without RLS) who received chronic HD treatment between April 2014 and April 2015. Demographic and biochemical data were evaluated in all patients, and statistical analyses were performed. Results The mean age and mean dialysis vintage of all patients (56% women) included in the study were 65.3 +/- 11.6 years and 41.5 +/- 36.5 months, respectively. Serum hepcidin, hemoglobin A1C (HbA1C), and ferritin levels were significantly higher in patients with RLS (p = 0.001,p = 0.032,p = 0.042, respectively). In addition, a positive correlation was found between International Restless Legs Syndrome Study Group severity scale score and serum hepcidin levels, HbA1C, and ferritin (r = 0.387,p = 0.001;r = 0.426,p = 0.034;r = 0.240,p = 0.046, respectively). A multivariate linear regression analysis revealed hepcidin and HbA1C to be independently associated with the presence of RLS. Conclusion A significant relationship was detected between RLS and increased serum hepcidin levels in chronic HD patients, and uncontrolled diabetes was noted to contribute to this association.