In humans and other primates, red blood cells (RBCs) constitutively express high levels of liver-type arginase 1 (Arg1), which regulates systemic l-arginine and nitric oxide (NO) bioavailability, particularly under pathological conditions such as sickle cell disease. In contrast, the role of RBC Arg1 in mice in vivo remains poorly defined. Here, we investigated the contribution of RBC Arg1 to systemic l-arginine metabolism, NO bioavailability, and cardioprotection following acute myocardial infarction in vivo. Comparative analyses of human blood fractions revealed that arginase activity in RBCs is comparable to that in white blood cells and is predominantly localized to the RBC membrane. In contrast, arginase activity in mouse RBC membranes was 13,500-fold lower as compared to human RBC membranes as measured by C-l-ornithine formation. To assess the in vivo relevance of RBC Arg1, we generated RBC-specific Arg1 knockout (KO) mice using the Cre/loxP technology. RBC Arg1 KO mice exhibited normal erythropoiesis and hematologic parameters. Moreover, systemic l-arginine and l-citrulline levels were preserved, while l-ornithine levels were lower in plasma of RBC Arg1 KO mice as compared to wildtype controls; whereas circulating NO metabolites, systemic hemodynamics, cardiac function, and infarct size post-acute myocardial infarction were preserved. These findings demonstrate that, unlike in humans, in mice RBC Arg1 plays a negligible role in regulating systemic l-arginine homeostasis and cardioprotection, underscoring critical interspecies differences and the need for human studies to evaluate the pathophysiological relevance of RBC arginase.