Fyn, an Src kinase family member, acts as a negative regulator of NF-E2-related factor 2 (Nrf2). Under stressful conditions, Nrf2 translocates into the nucleus and binds to the antioxidant response element (ARE), activating defensive gene expression. Once Nrf2 completes activation, Fyn phosphorylates tyrosine 568 of Nrf2, resulting in the nuclear export and degradation of Nrf2. The present studies demonstrate that within 0.5 h of antioxidant treatment in human hepatoblastoma (HepG2) cells, Fyn exports out of the nucleus, allowing Nrf2 unimpeded movement to the ARE. Mutation of tyrosine 213 of Fyn stymied nuclear export, suggesting that tyrosine phosphorylation controls nuclear export. Mass spectrometry confirmed tyrosine 213 as the site of phosphorylation. ChIP and real-time PCR assays revealed that FynY213A mutant caused decreased binding of Nrf2 to the promoter of defensive gene NAD(P)H:quinone oxidoreductase 1 (NQO1) and decreased NQO1 expression by 5-fold (P<0.0001) compared to wild-type Fyn. In addition, a putative nuclear export signal (NES) was identified, and mutation of it also inhibited nuclear export of Fyn. Furthermore, FynY213A caused an increased susceptibility to cell death following treatment with etoposide in mouse hepatoma (Hepa-1) cells. The preinduction regulation of Nrf2 is controlled by the nuclear export of Fyn, allowing for activation of defensive gene expression.