H₂O₂ is a key substance in the oxidative stress. To evaluate the antioxidant activity of intact human umbilical vein endothelial cells (HUVEC), we measured the H₂O₂ removal rate by the cell in monolayer culture at various H₂O₂ concentrations (1–300 μM). It was shown that the removal reaction can be divided into two kinetically different reactions: reaction 1 apparently following the Michaelis–Menten kinetics and reaction 2 following the first-order kinetics. Reaction 1, which was diminished by treatment with diethyl maleate, could be attributed to GPx. Reaction 2, which was inhibited by aminotriazole, was principally attributable to catalase, though non-enzymatic reactions may contribute to it partially. Furthermore, we have constructed a mathematical model for the H₂O₂ elimination including the pentose phosphate pathway enzymes, GSSG reductase and GSH peroxidase. On the basis of the known kinetics and observed activities of the enzymes, the model could reproduce well the observed concentration dependence of the H₂O₂ removal rate. It was suggested from the simulation study that GSSG reductase is more important than G6PD in determining the rate of the NADPH-dependent H₂O₂ elimination.