Activation of Nrf2 cascade can protect retinal pigment epithelium (RPE) cells from hydrogen peroxide (H2O2) and other oxidative injury. The current study identified microRNA-601 (miR-601) as a novel cullin 3 (Cul3)-targeting miRNA that activates Nrf2 cascade. In ARPE-19 cells and primary human RPE cells, forced overexpression of miR-601 significantly inhibited Cul3 3'-UTR activity and downregulated Cul3 mRNA/protein expression, leading to Nrf2 protein stabilization and its nuclear translocation as well as expression of anti-oxidant response elements (ARE)-dependent genes (HO1, NQO1 and GCLC). H2O2 treatment increased miR-601 levels in RPE cells. Significantly, ectopic miR-601 overexpression attenuated H2O2-induced oxidative injury and apoptosis in RPE cells. In contrast, miR-601 inhibition promoted Cul3 expression, lowered basal Nrf2 activation, and enhanced H2O2-induced oxidative stress and apoptosis in RPE cells. In ARPE-19 cells, CRISPC/Cas9-mediated knockout (KO) of Cul3 or Keapl not only mimicked, but also nullified, miR-601-inudced anti-H2O2 actions. Furthermore, Nrf2 silencing by targeted shRNAs abolished miR-601-inudced cytoprotection in H2O2-treated ARPE-19 cells. Taken together, we show that miR-601 activates Nrf2 signaling to protect RPE cells from H2O2 by targeting Cul3. (C) 2019 Elsevier Inc. All rights reserved.