Background: Hepcidin is a key regulator of iron homeostasis, while the clinical utility of hepcidin remains uncertain in hemodialysis (HD) patients. Objectives: Our study aimed to evaluate the predictive effect of serum hepcidin-25 on mortality in HD patients. Methods: A prospective observational cohort study of chronic HD patients were conducted at Xuzhou Central Hospital, Jiangsu, China, during years 2015 - 2017. The data on demographic factors, dialysis vintage, comorbidities, and laboratory measures were collected. Kaplan-Meier survival analysis was used to compare the effect of serum hepcidin-25 levels on mortality. Logistic regression models and multivariate Cox proportional hazard models were performed to identify the predictors of all-cause mortality in HD patients. Results: A total of 159 patients were included in this cohort, who were stratified into three groups by tertiles of hepcidin-25 values, and their 2-year overall mortality rate was 11.94%. The Kaplan-Meier analysis showed that patients with the highest tertile of serum hepcidin-25 had significantly higher all-cause mortality than in the two lower tertiles (P < 0.001). Serum hepcidin-25 was an independent risk factor for all-cause mortality after multivariate adjustments using logistic regression models and Cox proportional hazard models. Conclusions: A higher level of serum hepcidin-25 in chronic HD patients could be associated with increased mortality. Further studies are needed in a larger size of HD patients with a longer term of follow up.