() infection can lead to the occurrence of hypoxia, however, the underlying mechanisms have not been fully elucidated. β-hemolysin (Hlb) induced hemolysis of red blood cells (RBCs) requires a temperature transition from "hot" to "cold," a phenomenon not observed under physiological conditions. In this study, we discovered that RBCs treated with Hlb exhibited a high level of intracellular Ca and underwent a shape transformation from biconcave discoid to spherical, which was contingent upon the degradation of sphingomyelin of the cell membrane and led to impaired oxygen transport. The increase in intracellular Ca levels induced by Hlb was dependent on the activation of the ion channel N-methyl-D-aspartate receptor. Furthermore, we found that Hlb-induced Ca influx increased the cytoplasmic pH and subsequently attenuated the oxygen release from RBCs, which were also observed in both transgenic mice and a murine model with challenge. Our findings reveal a novel role for Hlb as sphingomyelinase in impairing RBC function under non-lytic conditions, shedding light on the mechanism behind hypoxia associated with infection.