Benzene is a chemical contaminant widespread in industrial and living environments. The oxidative metabolites of benzene induce toxicity involving oxidative damage. Protecting cells and cell membranes from oxidative damage, glucose-6-phosphate dehydrogenase (G6PD) maintains the reduced state of glutathione (GSH). This study aims to investigate whether the downregulation of G6PD in K562 cell line can influence the oxidative toxicity induced by 1,4-benzoquinone (BQ). G6PD was inhibited in K562 cell line transfected with the specific siRNA of G6PD gene. An empty vector was transfected in the control group. Results revealed that G6PD was significantly upregulated in the control cells and in the cells with inhibited G6PD after they were exposed to BQ. The NADPH/NADP and GSH/GSSG ratio were significantly lower in the cells with inhibited G6PD than in the control cells at the same BQ concentration. The relative reactive oxygen species (ROS) level and DNA oxidative damage were significantly increased in the cell line with inhibited G6PD. The apoptotic rate and G2 phase arrest were also significantly higher in the cells with inhibited G6PD and exposed to BQ than in the control cells. Our results suggested that G6PD inhibition could reduce GSH activity and alleviate oxidative damage. G6PD deficiency is also a possible susceptible risk factor of benzene exposure.