Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.