Oxidative stress is one of the elements causing aging and related diseases. Inhibiting Nrf2 activity or increasing oxidative pressure can replicate the deficits of premature aging. SIRT6 is one of the few proteins that can regulate both life span and aging. Deletion of SIRT6 in human cells impairs the antioxidant capacity of cells, which results in the accumulation of intracellular reactive oxygen species and DNA oxidation products. Characterization of the binding of Nrf2 with SIRT6 is critical for understanding the modulation of Nrf2-correlated cell activities by SIRT6. The yeast two-hybrid experiments showed that the binding of Nrf2 with SIRT6 is mediated by Neh1 and Neh3 domains. The elimination of the Neh1 and Neh3 domains decreased the binding stability and free energy, according to the molecular dynamic analysis. The roles of theses domains in mediating the binding were confirmed by co-immunoprecipitation. In cells transfected with the small interfering RNA (siRNA) targeting the Nrf2 Neh1 domain and plasmids overexpressing domain-mutant Nrf2, it was discovered that Nrf2 lost its activity to stimulate the transcription of antioxidant genes in the absence of Neh1 and Neh3 domains.