Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase-induced deletion of Rac1 on a Rac2 null genetic background. The Rac1(-/-);Rac2(-/-) mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1(-/-);Rac2(-/-) RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1(-/-);Rac2(-/-) erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1(-/-);Rac2(-/-) erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.