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Background: An individual with visceral Leishmaniasis (VL) commonly present with anemia and one of the VL treatment center in northwest Ethiopia has been recommended iron-folic acid supplementation to these patients. But there is no documented evidence whether iron-folic acid supplementation improves the hematological profile of patients. Therefore, the study aimed to assess change in hemoglobin (Hb) and its determinant factors among VL patients with and without iron-folic acid supplementation in northwest Ethiopia., Methods: A retrospective cohort study was conducted from January 2015 to December 2016. Data were entered into Epi-Data version 3.1 and transferred to Statistical Package for Social Science (SPSS) version 20 for analysis. Independent sample T-test and linear regression were used to compare the change in Hb and identify factors associated with a change in Hb, respectively. A 95% confidence level and p -values less than 0.05 were used determine statistically significant., Results: From a total of 602 VL patients, 299 (49.7%) were from University of Gondar hospital. The mean (±SD) change of Hb from baseline to end of treatment was 0.99(±1.64) and 1.61(±1.88) g/dl with and without iron-folate supplementation, respectively, with mean difference 0.62, 95% CI (0.34, 0.90) and a p -value of < 0.0001. In multiple linear regressions, combination therapy of sodium stibogluconate-paramomycin (SSG-PM) was positively associated with a change of Hb (β [SE, p]: 0.710/0.15, < 0.0001). Whereas age (- 0.030/0.009, 0.001), nasal bleeding (- 0.261/0.123, 0.035), baseline white blood cell (- 0.139/0.044, 0.002) and hemoglobin (- 0.513/0.031, < 0.0001), end of treatment spleen size (- 0.059/0.015, < 0.0001) and iron-folic acid supplementation (- 0.574/0.163, < 0.0001) were negatively associated with change of Hb., Conclusion: Iron-folic acid supplementation had a negative effect on the change of Hb. A combination therapy of SSG-PM, age, nasal bleeding, baseline white blood cells and Hb, and iron-folic acid supplementation were the determinants of change of Hb. Therefore, avoiding iron-folic acid supplementation and strengthening VL treatment with a combination of SSG-PM and, and early identification of complications is recommended for a better outcome., Competing Interests: Ethical approval for this study was obtained from the ethical review board of the University of Gondar. Permission letter was also obtained from the two treatment centers. In addition, personal identifiers were not retrieved and to maintain confidentiality and data were kept locked.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.