To study the hemolytic effect of polyphyllin II (PP II) mediated by anion channel protein and glucose transporter 1 (GLUT1), in order to initially reveal its hemolytic mechanism in vitro. In the experiment, the spectrophotometric method was adopted to detect the hemolysis of PP II in vitro and the effect of anion channel-related solution and blocker, glucose channel-related inhibitor and multi-target drugs dehydroepiandrosterone (DHEA) and diazepam on the hemolysis of PP II. The scanning electron microscope and transmission electron microscope were used to observe the effect of PP II on erythrocyte (RBC) morphology. The results showed that PP II -processed blood cells were severely deformed into spherocytes, acanthocyturia and vesicae. According to the results of the PP II hemolysis experiment in vitro, the anion hypertonic solution LiCl, NaHCO3, Na2SO4 and PBS significantly inhibited the hemolysis induced by PP II (P < 0.05), while blockers NPPB and DIDS remarkably promoted it (P < 0.01). Hyperosmotic sodium chloride, fructose and glucose at specific concentrations notably antagonized the hemolysis induced by PP II (P < 0.05). The glucose channel inhibitor Cytochalasin B and verapamil remarkably antagonized the hemolysis induced by PP II (P < 0.01). The hemolysis induced by PP II could also be antagonized by 1 gmol x L(1) diazepam and 100 μmol x L(-1) DHEA pretreated for 1 min (P < 0.01). In conclusion, the hemolytic mechanism of PP II in vitro may be related to the increase in intracellular osmotic pressure and rupture of erythrocytes by changing the anion channel transport activity, with GLUT1 as the major competitive interaction site.