Reactive molecules have diverse effects on cells and contribute to several pathological conditions. Cells have evolved complex protective systems to neutralize these molecules and restore redox homeostasis. Previously, we showed that association of nuclear factor (NF)-erythroid-derived 2 (E2)-related factor 2 (NRF2) with the nuclear matrix protein NRP/B was essential for the transcriptional activity of NRF2 target genes in tumor cells. The present study demonstrates the molecular mechanism by which NRP/B, via NRF2, modulates the transcriptional activity of antioxidant response element (ARE)-driven genes. NRP/B is localized in the nucleus of primary brain tissue and human neuroblastoma (SH-SY5Y) cells. Treatment with hydrogen peroxide (H(2)O(2)) enhances the nuclear colocalization of NRF2 and NRP/B and induces heme oxygenase 1 (HO1). Treatment of NRP/B or NRF2 knockdowns with H(2)O(2) induced apoptosis. Co-expression of NRF2 with members of the Kelch protein family, NRP/B, MAYVEN, or MAYVEN-related protein 2 (MRP2), revealed that the NRF2-NRP/B complex is important for the transcriptional activity of ARE-driven genes HO1 and NAD(P)H:quinine oxidoreductase 1 (NQO1). NRP/B interaction with Nrf2 was mapped to NRF2 ECH homology 4 (Neh4)/Neh5 regions of NRF2. NRP/B mutations that resulted in low binding affinity to NRF2 were unable to activate NRF2-modulated transcriptional activity of the ARE-driven genes, HO1 and NQO1. Thus, the interaction of NRP/B with the Neh4/Neh5 domains of NRF2 is indispensable for activation of NRF2-mediated ARE-driven antioxidant and detoxifying genes that confer cellular defense against oxidative stress-induced damage.