In the past, many in vitro hepatocyte injury models developed for liver regeneration used carbon tetrachloride as irritant chemical. Recently, carbon tetrachloride usage was prohibited due to serious deleterious effects to human and environment. There is an urgent need to develop a new acute chemical-induced hepatocyte injury model using other chemical compound to replace carbon tetrachloride. In this study, we used hydrogen peroxide (H2O2) to induced hepatocyte injury with HepG2 as the liver cell model. HepG2 injury was established by exposing the cells to CC50 of H2O2 at the concentration of 2.4 mM, predetermined via MTT assay for 2 h exposure. Aspartate aminotransferase (AST) activity was measured to determine the extent of cellular injury and quantitative PCR was carried out to determine the expression of inflammatory genes of the cells 24 h after H2O2 exposure. The results showed that AST activity increased with time and peak at 24 h after H2O2 exposure. Quantitative PCR analysis demonstrated that expression of inflammatory genes (TGF-β1, MMP-3, NF-κβ, IL-8 and IL-6) increased significantly. In addition, the gene expression of GPX, an anti-oxidant enzyme was also increased significantly in response to oxidative stress. In summary, H2O2 demonstrated excellent capability in inducing oxidative injury to HepG2 and together they represent an ideal acute chemical-induced injury model that can be used for liver regeneration study. Our results also provide input for inflammatory gene expression in the hepatocyte injury model.