Etavopivat is an investigational, oral, small molecule activator of erythrocyte pyruvate kinase (PKR) in development for the treatment of sickle cell disease (SCD) and other hemoglobinopathies. PKR activation is proposed to ameliorate the sickling of SCD red blood cells (RBC) through multiple mechanisms, including reduction of 2,3-diphosphoglycerate (2,3-DPG), which consequently increases hemoglobin (Hb)-oxygen affinity; increased binding of oxygen reduces HbS polymerization and sickling. In addition, PKR activation increases adenosine triphosphate (ATP) produced via glycolytic flux, which helps preserve membrane integrity and RBC deformability. We evaluated the pharmacodynamic response to etavopivat in non-human primates (NHP) and in healthy human subjects, and the effects in RBC from patients with SCD after ex vivo treatment with etavopivat. A single dose of etavopivat decreased 2,3-DPG in NHP and healthy subjects. Hb-oxygen affinity was significantly increased in healthy subjects after 24 hours. Following daily dosing of etavopivat over 5 consecutive days in NHP, ATP was increased by 38% from baseline. Etavopivat increased Hb-oxygen affinity and reduced sickling in RBC collected from SCD patients with either HbSS or HbSC disease. Collectively, these results demonstrate the ability of etavopivat to decrease 2,3-DPG and increase ATP, resulting in increased Hb-oxygen affinity and improved sickle RBC function. Etavopivat is currently being evaluated in clinical trials for the treatment of SCD. ClinicalTrials.gov identifier: NCT03815695 Significance Statement Etavopivat-a small molecule activator of the glycolytic enzyme erythrocyte pyruvate kinase -decreased 2,3-diphosphoglycerate in red blood cells (RBC) from non-human primates and healthy subjects and significantly increased hemoglobin (Hb)-oxygen affinity in healthy subjects. Using ex vivo RBC from donors with sickle cell disease (SCD) (HbSS or HbSC genotype), etavopivat increased Hb-oxygen affinity and reduced sickling under deoxygenation. Etavopivat shows promise as a treatment for SCD, that potentially might reduce vaso-occlusion and improve anemia.