Background: Diabetes mellitus, characterized by chronic hyperglycemia, is known to have a deleterious effect on erythrocyte structure and hemodynamic characteristics, which eventually contribute to diabetes-associated vascular complications. Protein kinase C alpha (PKCα) is a major regulator of many metabolic processes and structural changes in erythrocytes, and may play a significant role in the development of hyperglycemia-mediated cellular abnormalities. Aim: We hypothesized that acute hyperglycemic stress may affect erythrocyte structure and metabolic properties through its effect on PKCα membrane content and activity. Results: Erythrocytes, from healthy individuals acutely exposed to a glucose enriched media, showed a significant decrease in the membranous fraction of PKCα and its phosphorylation (p = 0.005 and p = 0.0004, respectively). These alterations correlated with decreased affinity of PKCα to its membrane substrates (4.1R and GLUT1) and reduced RBC deformability (p = 0.017). Pre-activation of erythrocytes with PKC activator, PMA, minimized the effect of glucose on the membrane PKCα fraction and RBC deformability (p > 0.05). Conclusion: Acute glycemia-induced inhibition of PKCα membranous translocation and activation is associated with reduced erythrocyte membrane deformability.