The Sestrin2 (Sesn2) gene encodes a conserved antioxidant protein that is induced on oxidative stress and protects cells against reactive oxygen species. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates expression of a variety of antioxidant genes via binding to the antioxidant-response element (ARE), but the role of Nrf2 in Sesn2 gene expression has not been elucidated yet. The present study investigated whether the Nrf2-ARE pathway regulates Sesn2 gene expression and the identification of the molecular mechanism. The Nrf2 activators, tert-butylhydroquinone (t-BHQ) and sulforaphane (SFN), up-regulated Sesn2 expression in a dose- and time-dependent manner in hepatocytes. Also, t-BHQ increased Sesn2 mRNA and luciferase gene activity, whereas the levels of Sesn1 and Sesn3 mRNA were not affected by t-BHQ treatment. The specific role of Nrf2 in Sesn2 induction was verified by using Nrf2 overexpression plasmid and Nrf2 knockout or knockdown cells. In silico analysis of the 5' upstream region of Sesn2 gene identified a putative ARE sequence. Deletion of the putative ARE demonstrated that the ARE from -550 to -539 bp in the human Sesn2 promoter was critical for the Nrf2-mediated response. Moreover, SFN injection increased Sesn2 mRNA and protein levels in the livers of mice. Knockdown experiments with Sesn2 siRNA showed that Sesn2 is required for the Nrf2-mediated cytoprotective activity against hydrogen peroxide. Our results suggest that the Nrf2-ARE pathway is critical for Sesn2 gene expression and might protect against oxidative stress.