Gardos channel, the erythrocyte Ca²⁺-activated K⁺ channel (K_Ca3.1), is considered a major regulator of red blood cell (RBC) volume by mediating efflux of potassium and thus cell dehydration and shrinkage. However, the functional importance of K_Ca3.1 in RBC in vivo is incompletely understood. Here, we used K_Ca3.1^−/−-mice to investigate the consequences of K_Ca3.1 deficiency for RBC indices, functions, and sequestration. RBCs of K_Ca3.1^−/−-mice of all ages were mildly macrocytic but their biconcave appearance being preserved. RBC number, total hemoglobin, and hematocrit were unchanged in the adult K_Ca3.1^−/−-mice and increased in the premature K_Ca3.1^−/−-mice. Filterability, Ca²⁺-dependent volume decrease and osmotic tolerance of RBCs lacking K_Ca3.1 were noticeably reduced when compared to RBC of wild-type littermates. Deformability to increasing shear stress was unchanged. Strikingly, K_Ca3.1^−/−-mice developed progressive splenomegaly which was considerable (∼200% of controls) in the >6-month-old mice and was paralleled by increased iron deposition in the aged mice presumably as a consequence of enhanced RBC sequestration. Daily injections of the K_Ca3.1-blocker TRAM-34 (120 mg/kg) also produced mild splenomegaly in wild-type mice. We conclude that genetic deficit of erythroid K_Ca3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly.