Background: Anemia is an indirect cause of high maternal mortality rates worldwide characterized by low levels of hemoglobin, hepcidin, and serum ferritin. Hepcidin is a biomarker of iron metabolism in the body while ferritin acts as a store of iron. Hepcidin and ferritin levels during pregnancy will decrease. To restore the levels, supplementation is required either orally or intravenously. This study aimed to determine the effect of oral and intravenous iron supplementation on hepcidin and ferritin levels. Methods: This was an experimental laboratory study using Posttest Only Control Group Design. The sample was 24 Rattus Norvegicus divided into 4 groups: the negative control group without treatment, the positive control group given NaNo, the, P1 given oral iron supplementation, and the P2 given intravenous iron. The maintenance of the test animals in this study was carried out at the Pharmacy laboratory of Universitas Andalas Padang in June-August 2021. The experimental animals were Rattus Norvegicus Wistar females weighing 200-250 grams, aged between 3-4 months conditioned to be pregnant. The serum examination of hepcidin and ferritin levels used the ELISA method and the normality test used Shapiro Wilk. Then, the significance test used One-Way ANNOVA followed by multiple comparisons of Bonferroni types. Results: The results showed that there were significant differences (p<0.05) in hepcidin levels in the positive control group, treatment group 1, treatment 2 that were 219.52 ng/ml, 220.27 ng/ml, and 221.49 ng/ml. Likewise, the ferritin levels in the positive control group, treatment 1, and treatment 2 were 5.91 ng/ml, 6.81 ng/ml and 7.72 ng/ml. Conclusion: Based on the findings, it can be concluded that oral and intravenous iron supplementation had an effect on increasing serum hepcidin and ferritin levels in Rattus norvegicus pregnant strain Wistar with anemia. Keywords: Oral iron supplementation, intravenous supplementation, hepcidin and ferritin