beta-thalassemia (beta-thal) is a worldwide hereditary red cell disorder characterized by severe chronic anemia. Recently, the pyruvate kinase (PK) activator mitapivat has been shown to improve anemia and ineffective erythropoiesis in a mouse model of beta-thal and in non-transfusion dependent thalassemic patients. Here, we showed that in vitro CD34+ derived erythroblasts from beta-thal (codb039) patients are characterized by persistent expression of two PK isoforms, PKR and PKM2, when compared to healthy cells. Activation of PKR and PKM2 via mitapivat promoted a significant metabolic reprogramming of beta-thal erythroblasts, resulting in higher levels of high-energy phosphate compounds, including adenosine triphosphate (ATP) and triphosphate nucleoside pools. Proteomics analyses revealed an accumulation of PKR, suggesting a possible beneficial effect of mitapivat on the stability of PKs. Increased ATP availability was accompanied by a higher degree of protein phosphorylation, especially of proteins involved in cell cycle regulation at the transcriptional, translational, and post-translational levels, supporting the effect of mitapivat on erythroid maturation. Upon treatment with mitapivat, beta-thal erythroblasts showed decreased markers of oxidation, including cysteine oxidative post-translational modifications, down-regulation of HSP-70 and Prdx2 expression, and normalization of the redox-dependent sub-cellular distribution of the latter enzyme. Collectively, our data support a protective effect of mitapivat in beta-thal erythropoiesis, an effect favored by its activation of persistently expressed PKR and PKM2. In addition to the anticipated benefits on energy metabolism, we report that mitapivat treatment mitigated the oxidative damage in beta-thal erythropoiesis, ensuring improved beta-thal erythroblast maturation and survival.