Intro: Sudden cardiac death (SCD) from ventricular arrhythmias (VT/VF) claims more lives each year than all other disease-related causes of death combined. The underlying links between HF and SCD remain unclear. Reactive oxygen species (ROS) are central to SCD pathophysiology, but their effects depend on where and how ROS are generated. Employing innovative approaches, we hypothesized that mitochondrial ROS (mROS) is a principal source of oxidative stress in HF and in vivo reduction of mROS mitigates SCD. Methods: In a unique guinea pig model that mimics human non-ischemic HF with a high incidence of SCD, we performed a randomized 2x2 crossover study with MitoTEMPO therapy. We employed novel, genetically encoded ratiometric redox sensors for quantitative measurement of cytosol (cROS) or mROS of cardiomyocytes. Results: We show for the first time that mROS is elevated in failing hearts and that a mitochondrially targeted antioxidant (MitoTEMPO) normalizes cellular ROS, while NADPH oxidase inhibition is effective in correcting only the cROS component. in vivo mitoTEMPO treatment can prevent or reverse HF while eliminating SCD by decreasing T-wave dispersion and suppressing premature ventricular complexes (PVCs). Scavenging mROS suppressed chronic HF-induced remodeling of the expression proteome and prevented specific phosphoproteome alterations. Pathway analysis of mROS-sensitive networks indicated that, in HF, increased mROS disrupts the normal coupling between cytosolic signals and the nuclear gene programs driving mitochondrial function, antioxidant enzymes, Ca2+ handling and action potential repolarization. Conclusions: mROS drive both acute emergent events, such as electrical instability leading to SCD, and mediates chronic HF remodeling, characterized by suppression or altered phosphorylation of metabolic, antioxidant and ion transport protein networks. Impaired mROS scavenging cause failing hearts to become more vulnerable to demand-induced oxidative stress and promote arrhythmic SCD. Maintaining global redox balance by enhanced mROS scavenging may be an effective strategy for SCD therapy in HF.