Reactive thiols of proteinaceous cysteines are vital to cell biology by serving as sensor, effector and buffer of environmental redox fluctuations. Being the major source as well as the prime target of reactive oxygen species (ROS), the mitochondrion confronts great challenges in preserving its thiol pool. Here we identify ROS modulator 1 (ROMO1), a small inner mitochondrial membrane protein, as a master thiol-protector of the mitochondrial cysteinome. Being redox sensitive and reactive, ROMO1 shields functional thiols by scavenging ROS and preventing irreversible thiol oxidations. Such ROMO1-mediated thiol protection exerts extensively beneficial effects on mitochondria, such as promoting energy metabolism and Ca2+ uniport while inhibiting vicious membrane permeability transition. Importantly, ROMO1 reverses mitochondrial cysteinome oxidations in multiple organs and retards their functional declines in aged male mice. These findings unravel an exquisite thiol-protection mechanism of the mitochondrial cysteinome, and mark ROMO1 as a potential target for combating oxidative stress and improving healthspan.