Although the therapeutic efficacy of β(654)-thalassaemia treatment using a combination of RNAi and antisense RNA to balance the synthesis of α- and β-globin chains has been demonstrated previously, and the safety of lentiviral delivery remains unclear. Herein, we used the same β(654)-thalassaemia mouse model to develop a therapy involving direct delivery of siRNA and antisense RNA plasmids via intravenous injection to simultaneously knock down α-globin transcript levels and restore correct β-globin splicing. The amount of α-globin mRNAs in siRNA-treated MEL cells decreased significantly, and the properly spliced β-globin mRNA was restored in HeLaβ(654) cells transfected with pcDNA-antisense plasmid. Furthermore, treatment of β(654)-thalassaemic mice with siRNA and antisense RNA plasmids resulted in significant reduction of poikilocytosis and reticulocyte counts in blood samples, decreased nucleated cell populations in bone marrow, and reduced intrasinusoidal extramedullary haematopoiesis loci and iron accumulation in liver. RT-PCR analysis revealed that treatment resulted in down-regulation of α-globin mRNA synthesis by ~50% along with an increase in the presence of normally spliced β-globin transcripts, indicating that the phenotypic changes observed in β(654)-thalassaemic mice following treatment resulted from restoration of the balance of α/β-globin biosynthesis.