Although the introduction of erythropoietin-stimulating agents (ESAs) has led to better clinical outcomes in patients undergoing hemodialysis (HD), fluctuations in hemoglobin (Hb) levels, known as Hb variability, are frequently observed. However, only a few studies have evaluated the association between Hb variability and nutritional status in patients undergoing HD. Methods: In this prospective study conducted between March 1, 2020, and June 1, 2022, we included 109 patients aged over 20 years undergoing HD and receiving darbepoetin. We checked the average NESP (darbepoetin-alfa; Kyowa Kirin Korea Co., Ltd.) dose weekly and nutritional parameters such as body mass index (BMI), fat tissue index (FTI), and lean tissue index obtained by body composition monitoring. Additionally, the ESA resistance index (ERI) and the coefficient of variation of Hb (Hb-CV) were evaluated. Results: In this study, the mean age of the patients was 64.0 ± 11.9 years, and 55.0% were male. Mean Hb was 10.7 ± 1.3 g/dL. Patients were categorized into three groups according to the ERI or Hb-CV tertiles. The highest ERI tertile was associated with lower Hb levels, BMI, and FTI. The highest Hb-CV tertile was associated with lower BMI and FTI. In multiple linear regression analysis, FTI was negatively associated with ERI (β = –0.218, p = 0.01) and Hb-CV (β = –0.181, p = 0.04). Conclusion: These findings suggest that FTI is negatively associated with ERI and Hb-CV, and that ESAs responsiveness and Hb variability are associated with FTI in patients undergoing HD with darbepoetin treatment.