Diamond-Blackfan anemia (DBA) is a ribosomopathy encompassed within bone marrow failure (BMF) syndromes primarily characterized by erythroblastopenia. Additionally, increased incidence of cancer and congenital abnormalities have been reported. Allogenic hematopoietic stem cell transplantation (HSCT) currently represents the only curative treatment for BMF in DBA patients. However, its associated mortality and morbidity demand new therapies. In DBA, the most commonly mutated gene is RPS19 (25%). Therefore, we focused our studies on the preclinical development of an ex-vivo gene therapy approach to correct the genetic defect of RPS19-deficient cells using a therapeutic lentiviral vector (LV) harbouring a codon-optimized sequence of RPS19-cDNA (PGK.CoRPS19.Wpre*-LV) developed in our laboratory. Therapeutic efficacy of the LV was evaluated in bone marrow (BM)-CD34⁺ cells from RPS19-deficient patients. Transduction of these CD34⁺ cells with the LV significantly increased the number of BFU-E colonies and reverted the red blood cell differentiation defect characteristic of DBA-CD34⁺ cells, as revealed by the increased output of CD71⁺/CD235⁺ mature erythroid cells both in vitro and in vivo. Remarkably, DBA-CD34⁺ cells transduced with the LV were also capable of restoring the hematopoiesis of immunodeficient mice. In these studies we also confirmed the healthy status of transplanted immunodeficient recipients, as well as the polyclonal repopulation pattern of corrected hematopoietic cells. In conclusion, the preclinical studies conducted in this work indicate that the LV-mediated gene therapy with the PGK.CoRPS19-LV, for which the Orphan drug designation “EMA/OD/0000060656” has already been obtained, should provide an efficient and safe approach to restore the hematological defects characteristic of RPS19-deficient DBA patients.