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Objective: To explore and analyze the reducing hemolysis effects of PEGylated puerarin on G6PD-deficient rat erythrocytes.Methods The rat model with G6PD-deficiency was established via injecting 1% acetylphenylhydrazine into the subcutaneous part of nuchal region.Then the G6PD-deficient erythrocyte suspension obtained from this rat model was used to evaluate the hemolytic effects of puerarin (PUE) and PEGylated puerarin (PEG-PUE) via hemolytic activity and erythrocyte osmotic fragility assay.Results It was found that PUE could cause a serious hemolysis to the erythrocyte suspension, and with the increase of drug concentration and the prolongation of drug incubation time, the hemolysis rate of PUE is up to 40%.While the addition of PEG-PUE to the erythrocyte suspension revealed no significant hemolysis.Additionally, the result of erythrocyte osmotic fragility indicated that PEG-PUE exerted a slight effect on the erythrocyte membranes, and its H50% value (32 mM) was about one-third of that of PUE.Conclusion These results demonstrated that PEG-PUE could play a significant role in reducing the hemolytic side effect of PUE.The low hemolytic activity of PEG-PUE makes it favorable candidate for in vivo tests and could also provide useful insight for further formulation development as an innovative drug.