β-Thalassaemia is an inherited red blood cell disorder characterised by diminished or absent β-globin chain synthesis leading to ineffective erythropoiesis and anaemia. The failures of erythropoiesis and chronic anaemia lead to abnormal iron metabolism and systemic iron overload, contributing to cardiac, hepatic and endocrine dysfunction. Several studies have identified the hepatic peptide hepcidin (the master regulator of iron homeostasis) to be suppressed in β‑thalassaemia contributing to increased gastrointestinal iron absorption and iron retention by the reticular endothelia system. Hepcidin is under the negative control of the transmembrane serine protease 6 (TMPRSS6) via the cleavage of haemojuvelin (HJV), a co-receptor for the BMP-SMAD signalling pathway. Considering the central role of the hepcidin/HJV/TMPRSS6 axis in iron homeostasis, inhibition of TMPRSS6 expression increases hepcidin production and ameliorate anaemia in β-thalassaemia in mice. In this study, we investigated RNAi-mediated reduction of TMPRSS6 in β-thalassaemia (Hbbth3/+) mice using a liver-specific delivery with an optimised GalNac-conjugated siRNA targeting TMPRSS6 (SLN124). Two s.c doses of SLN124 (3mg/kg) in Hbbth3/+ mice over 35 days, was sufficient to normalise hepcidin expression and significantly improve red cell indices, as shown by an increase in haematocrit level, and reduction in reticulocyte counts, red cell distribution width and reactive oxygen species (ROS). We also observed a significant improvement in erythroid maturation and erythrocyte production, which was associated with a reduction in spleen size and regression of cardiac hypertrophy. Collectively, we show that SLN124 is able to ameliorate multiple clinical symptoms associated with chronic anaemia in a mouse model for β‑thalassaemia intermediate. The liver-specific reduction of TMPRSS6 expression using SLN124 represents a promising pharmacologic modality for the treatment of β‑thalassaemia, and potentially other disorders associated with ineffective erythropoiesis and iron overload.