The ubiquitous toxic metalloid arsenic (As) elicits pleiotropic adverse and adaptive responses in mammalian species. The biological targets of As are largely unknown at present. We analyzed the signaling pathway for induction of detoxification gene NAD(P)H:quinone oxidoreductase (Nqo1) by As. Genetic and biochemical evidence revealed that induction required CNC bZip transcription factor Nrf2 and the antioxidant response element (ARE) of Nqo1. As stabilized Nrf2 protein extending the t1/2 of Nrf2 from 21 to 200 min by inhibiting the Keap1/Cul3-depedent ubiquitination and proteasomal turnover of Nrf2. As markedly inhibited the ubiquitination of Nrf2, but did not disrupt the Nrf2/Keap1/Cul3 association in the cytoplasm. In the nucleus, As, but not phenolic antioxidant tBHQ, dissociated Nrf2 from Keap1 and Cul3, followed by dimerization of Nrf2 with a Maf protein (G/K). Chromatin immunoprecipitation demonstrated that Nrf2 and Maf associated with the endogenous Nqo1 ARE enhancer constitutively. As substantially increased the ARE occupancy by Nrf2 and Maf. In addition, Keap1 was shown to be ubiquitinated in the cytoplasm and deubiquitinated in the nucleus in the presence of As without changing the protein level, implicating nuclear-cytoplasmic recycling of Keap1. Our data reveal that As activates the Nrf2/Keap1 signaling pathway through a distinct mechanism from that by antioxidants, and suggest an "on-switch" model of Nqo1 transcription in which the binding of Nrf2/Maf to ARE controls both the basal and inducible expression of Nqo1.