Targeting redox vulnerability of cancer cells by pro-oxidants capable of generating reactive oxygen species (ROS) has surfaced as an important anticancer strategy. Due to the intrinsic narrow therapeutic window and other dangerous side effects of ROS generation, it is highly needed and challenging to develop pro-oxidative anticancer agents (PAAs) with high selectivity for generating ROS in cancer cells. Herein we report a hydrogen peroxide (H2O2)-activated Cu(II) pro-ionophore strategy to develop naphthazarin (Nap) as such type of PAAs based on the H2O2-mediated conversion of boronate to free phenol. The boronate-protected Nap (PNap) can exploit increased levels of H2O2 in HepG2 cells to in situ release Nap followed by its efflux via conjugation with reduced glutathione (GSH), allowing that the Nap-GSH adduct works as a Cu(II) ionophore to induce continuously GSH depletion via a reduction-dependent releasing of Cu(I) by GSH. This strategy endows PNap with the unprecedented ability to hit multi-redox characteristics (increased levels of H2O2, GSH and copper) of HepG2 cells, leading to ROS generation preferentially in HepG2 cells along with their selective death. Display omitted •A H2O2-activated Cu(II) pro-ionophore strategy was first used for developing pro-oxidative anticancer agent.•The protected naphthazarin (PNap) can be selectively activated by H2O2 in HepG2 cells.•The strategy allows PNap with the ability to hit multi-redox characteristics of HepG2 cells.•PNap plus Cu(II) induced selective ROS generation in HepG2 cells.•Mechanisms by which PNap plus Cu(II) killed HepG2 cells selectively were described.