Inappropriately low expression of the key iron regulator hepcidin (HAMP) causes iron overload in untransfused patients affected by β-thalassemia intermedia and Hamp modulation provides improvement of the thalassemic phenotype of the Hbb(th3/+) mouse. HAMP expression is activated by iron through the bone morphogenetic protein (BMP)-son of mothers against decapentaplegic signaling pathway and inhibited by ineffective erythropoiesis through an unknown "erythroid regulator." The BMP pathway is inactivated by the serine protease TMPRSS6 that cleaves the BMP coreceptor hemojuvelin. Here, we show that homozygous loss of Tmprss6 in Hbb(th3/+) mice improves anemia and reduces ineffective erythropoiesis, splenomegaly, and iron loading. All these effects are mediated by Hamp up-regulation, which inhibits iron absorption and recycling. Because Hbb(th3/+) mice lacking Tmprss6 show residual ineffective erythropoiesis, our results indicate that Tmprss6 is essential for Hamp inhibition by the erythroid regulator. We also obtained partial correction of the phenotype in Tmprss6 haploinsufficient Hbb(th3/+) male but not female mice and showed that the observed sex difference reflects an unequal balance between iron and erythropoiesis-mediated Hamp regulation. Our study indicates that preventing iron overload improves β-thalassemia and strengthens the essential role of Tmprss6 for Hamp suppression, providing a proof of concept that Tmprss6 manipulation can offer a novel therapeutic option in this condition.