Background <p>Hepcidin-25 plays an important role in regulating iron metabolism; however, the association between hepcidin-25 levels and hyporesponsiveness to erythropoiesis-stimulating agents (ESAs) is controversial. We aimed to clarify the associations between serum hepcidin-25 levels and hyporesponsiveness to ESAs in Japanese patients receiving hemodialysis, and between hepcidin-25 levels and other factors.</p> Methods <p>This observational cross-sectional study included hemodialysis patients recruited at Heisei-Hidaka Clinic in Japan from August 2023 to June 2024. Serum hepcidin-25 levels were measured by latex immunoassay. Hyporesponsiveness to ESAs was determined by the ESA resistance index (ERI). The correlation between hepcidin-25 levels and ERI was evaluated using Pearson’s correlation coefficient. We also investigated the patient characteristics associated with hepcidin-25 levels using multiple regression analysis.</p> Results <p>Hepcidin-25 levels were significantly negatively correlated with ERI (<i>r</i> = − 0.438, <i>p</i> = 0.0005). Hepcidin-25 levels also showed significant positive correlations with serum iron, transferrin saturation (TSAT), serum ferritin, and high sensitive C-reactive protein (hs-CRP), and significant negative correlations with hematocrit, unsaturated iron-binding capacity, total iron-binding capacity, and serum erythropoietin levels. Hepcidin-25 levels were significantly higher in the patients who received oral iron-containing preparations than in those without these preparations. Multiple regression analysis showed significant partial regression coefficients for ERI, hematocrit, TSAT, serum ferritin, hs-CRP, and the administration of oral iron-containing preparations.</p> Conclusion <p>Serum hepcidin-25 levels were significantly negatively correlated with the ERI. The results suggest that hepcidin-25 levels might be associated with ERI, hematocrit, TSAT, serum ferritin, hs-CRP, and the administration of oral iron-containing preparations.</p>