Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables, has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, antihypercholesterolemic, and anticarcinogenic actions. However, the cytoprotection and mechanism of hesperidin to neutralize oxidative stress in human hepatic L02 cells remain unclear. In this work, we assessed the capability of hesperidin to attenuate hydrogen peroxide (H(2)O(2))-induced cell damage by augmenting the cellular antioxidant defense. Real-time quantitative polymerase chain reaction, Western blot, and enzyme activity assay demonstrated that hesperidin upregulated heme oxygenase-1 (HO-1) expression to protect hepatocytes against oxidative stress. In addition, hesperidin also promoted nuclear translocation of nuclear factor erythroid 2-related factor (Nrf2). What's more, hesperidin exhibited activation of extracellular signal-regulated protein kinase 1/2 (ERK1/2). Besides, ERK1/2 inhibitor significantly inhibited hesperidin-mediated HO-1 upregulation and Nrf2 nuclear translocation. Taken together, the above findings suggested that hesperidin augmented cellular antioxidant defense capacity through the induction of HO-1 via ERK/Nrf2 signaling. Therefore, hesperidin has potential as a therapeutic agent in the treatment of oxidative stress-related hepatocyte injury and liver dysfunctions.