Despite its transport by glucose transporters (GLUTs) in vitro, it is unknown whether dehydroascorbic acid (oxidized vitamin C, DHA) has any in vivo function. To investigate, we created a chemical transport knockout model using the vitamin C analog 6-bromo-ascorbate. This analog is transported on sodium-dependent vitamin C transporters but its oxidized form, 6-bromo-dehydroascorbic acid, is not transported by GLUTs. Mice (gulo−/−) unable to synthesize ascorbate (vitamin C) were raised on 6-bromo-ascorbate. Despite normal survival, centrifugation of blood produced hemolysis secondary to near absence of red blood cell (RBC) ascorbate/6-bromo-ascorbate. Key findings with clinical implications were that RBCs in vitro transported dehydroascorbic acid but not bromo-dehydroascorbic acid; RBC ascorbate in vivo was obtained only via DHA transport; ascorbate via DHA transport in vivo was necessary for RBC structural integrity; and internal RBC ascorbate was essential to maintain ascorbate plasma concentrations in vitro/in vivo.