Hydra is a freshwater cnidarian polyp that regenerates any missing part after amputation. This study focuses on the potential role of injury-induced ROS signaling in triggering head regeneration. We detected immediately after mid-gastric bisection symmetrical levels of mitochondrial superoxide and asymmetrical levels of hydrogen peroxide (H2O2), higher in head-regenerating tips than in basal-regenerating ones. This asymmetry likely results from a higher superoxide dismutase (SOD) activity in head-regenerating tips while catalase is lower. Pharmacological treatments (Tiron, DPI) and transient gene silencing approaches (sod-1, catalase) indicate that signaling via mitochondrial ROS plays a role in wound healing while high levels of H2O2 are necessary for apical regeneration. Through paracrine signalling, H2O2 triggers CREB phosphorylation as well as death of interstitial cells, while H2O2 levels are amplified by apoptotic cells via a feedback loop. Thus, asymmetric ROS signaling immediately after bisection is critical to induce cell death and apical regeneration.