Mutations in HFE lead to hereditary hemochromatosis (HH) because of inappropriately high iron uptake from the diet resulting from decreased hepatic expression of the iron-regulatory hormone hepcidin. β-thalassemia is a congenital anemia caused by partial or complete loss of β-globin synthesis causing ineffective erythropoiesis, anemia, decreased hepcidin production, and secondary iron overload. Tmprss6 is postulated to regulate hepcidin production by cleaving Hemojuvelin (Hjv), a key modulator of hepcidin expression, from the hepatocyte surface. On this basis, we hypothesized that treatment of mouse models of HH (Hfe^−/−) and β-thalassemia intermedia (Hbb^th3/+) with Tmprss6 siRNA formulated in lipid nanoparticles (LNPs) that are preferentially taken up by the liver would increase hepcidin expression and lessen the iron loading in both models. In the present study, we demonstrate that LNP-Tmprss6 siRNA treatment of Hfe^−/− and Hbb^th3/+ mice induces hepcidin and diminishes tissue and serum iron levels. Furthermore, LNP-Tmprss6 siRNA treatment of Hbb^th3/+ mice substantially improved the anemia by altering RBC survival and ineffective erythropoiesis. Our results indicate that pharmacologic manipulation of Tmprss6 with RNAi therapeutics isa practical approach to treating iron overload diseases associated with diminished hepcidin expression and may have efficacy in modifying disease-associated morbidities of β-thalassemia intermedia.