Non-alcoholic fatty liver disease (NAFLD) has become the world’s most common liver disease. The disease can develop liver fibrosis or even carcinomas from the initial hepatic steatosis, and this process is influenced by many factors. Reactive oxygen species (ROS), as potent oxidants in cells, have been reported previously to play an important role in the development of NAFLD progression via promoting neutral lipid accumulation. Here, we found that ROS can promote lipid droplet formation in hepatocytes by promoting perilipin2 (PLIN2) expression. First, we used different concentrations of hydrogen peroxide to treat HepG2 cells and found that the number of lipid droplets in the cells increased, however also that this effect was dose-independent. Then, the mRNA level of several lipid droplet-associated genes was detected with hydrogen peroxide treatment and the expression of PLIN2, PLIN5, and FSP27 genes was significantly up-regulated (p < 0.05). We overexpressed PLIN2 in HepG2 cells and found that the lipid droplets in the cells were markedly increased. Interference with PLIN2 inhibits ROS-induced lipid droplet formation, revealing that PLIN2 is a critical factor in this process. We subsequently analyzed the regulatory pathway and protein interaction network that is involved in PLIN2 and found that PLIN2 can regulate intracellular lipid metabolism through the PPARα/RXRA and CREB/CREBBP signaling pathways. The majority of the data indicated the correlation between hydrogen peroxide-induced PLIN2 and lipid droplet upregulation. In conclusion, ROS up-regulates the expression of PLIN2 in hepatocytes, whereas PLIN2 promotes the formation of lipid droplets resulting in lipid accumulation in liver tissues.