Insights into the role of Ankyrin-1 (Ank-1) in the formation and stabilization of the red cell cytoskeleton have come from studies on the nb/nb mice, which carry hypomorphic alleles of Ank-1. Here, we revise several paradigms established in the nb/nb mice through analysis of an ENU-induced Ank-1-null mouse. Mice homozygous for the Ank-1 mutation are profoundly anemic in utero, and most die perinatally, indicating that Ank-1 plays a non-redundant role in erythroid development. The surviving pups exhibit features of severe hereditary spherocytosis (HS), with marked hemolysis, jaundice, compensatory extramedullary erythropoiesis, and tissue iron overload. Red cell membrane analysis reveals a complete loss of ANK-1 protein, and a marked reduction in beta-spectrin. As a consequence, the red cells exhibit total disruption of cytoskeletal architecture, and severely altered hemorheological properties. Heterozygous mutant mice, which have wild type levels of ANK-1 and spectrin in their RBC membranes, and normal red cell survival and ultrastructure, exhibit profound resistance to malaria, which is not due to impaired parasite entry into RBC. These findings provide novel insights into the role of Ank-1, and define an ideal model for the study of HS and malarial resistance.