Pyruvate kinase (PK) is a key enzyme involved in the final step of glycolysis, essential to produce adenosine triphosphate (ATP). Relatively decreased red blood cell (RBC) PK activity (reflected by a lower PK/hexokinase [HK] ratio) and PK thermostability (PK activity after exposure to heat) were recently identified as pathophysiological features of sickle cell disease (SCD). In this study, we investigated whether impaired PK function is associated with sickle RBC properties and SCD-related clinical manifestations. This study included 97 non-transfused patients with SCD (88 HbSS, 9 HbS/β0 thalassemia). PK thermostability was correlated with RBC parameters such as reticulocyte count (r = -0.402, p <
0.0001) and hemoglobin F (r = 0.394, p <
0.0001), and indicators of impaired functional properties of sickle RBCs, such as the point of sickling (r = -0.417, p <
0.0001), oxygen affinity (r = 0.408, p <
0.001) and RBC adhesion to laminin (r = -0.322, p = 0.024). Additionally, a low PK/HK ratio correlated with decreased PK thermostability (r = 0.308, p = 0.002), decreased RBC deformability (r = 0.268, p = 0.009), and elevated 2,3-diphosphoglycerate levels (r = -0.244, p = 0.016). Multivariate Poisson regression analysis demonstrated that reduced PK thermostability and PK/HK ratio were associated with a higher incidence of SCD-related clinical complications. For every 10-unit decrease in PK thermostability and 1-unit decrease in PK/HK ratio, the incidence of SCD-related clinical complications increased by 11% (p = 0.012) and 10% (p = 0.019), respectively. Altogether, these findings indicate that impaired PK function is related to compromised sickle RBC properties and SCD-related clinical manifestations. This supports the relevance and underlines the potential of PK activation therapy.