Objective: To study the effects of Arsenic trioxide (As₂O₃) on the toxicity and energy metabolism of RBCs. Methods: Firstly, RBCs were incubated with As₂O₃ at the final concentration of 0.2 mmol/L, 0.4 mmol/L, 0.8 mmol/L,1.6 mmol/L and 3.2 mmol/L in 37℃ incubator for 48 h, and then the cell viability and hemolysis rate of RBCs were measured by an enzyme labeling instrument. Finally,RBCs were incubated with As₂O₃ at a final concentration of 2.5 mmol/L for 5 h, and the relative quantitative analysis of targeted energy metabolites in RBCs was performed by liquid chromatography-tandem mass spectrometry. Results: As₂O₃ at final concentration of 0.2 mmol/L could improve the cell viability of RBCs, and the difference has statistically significant(P＜0.05), 0.4 mmol/L and 0.8 mmol/L As₂O₃ had no significant effect on the viability of RBCs, but high concentration (1.6 mmol/L and 3.2 mmol/L) As₂O₃ significantly reduced the viability of RBCs, and the difference has statistically significant(P＜0.05). The half maximal inhibitory concentration (IC₅₀) of As₂O₃ RBCs was 5 202 μmol/L. The hemolysis rate of RBCs increased gradually with the increasing concentration of As₂O₃, but low As₂O₃ concentration (0.2 mmol/L and 0.4 mmol/L) had little effect on the hemolysis rate, and the hemolysis rate at 0.8 mmol/L, 1.6 mmol/L and 3.2 mmol/L groups were increased gradually(P＜0.05), but the hemolysis rate was less than 6 %, and destruction of seemed not very serious. The results of energy metabolites showed that the contents of adenosine triphosphate (ATP), pyruvate(PA), and nicotinamide adenine dinucleotide phosphate (NADPH) were increased significantly in RBCs after incubated with 2.5 mmol/L As₂O₃ at 37℃ for 5 h, and the difference had statistically significant (P＜0.05). Conclusion: Low concentration of As₂O₃ may protect RBCs by promoting glycolysis and pentose phosphate pathway.