It is well established that oxidative stress is an important cause of cell damage associated with the initiation and progression of many diseases. Consequently, all air-living organisms contain antioxidant enzymes that limit oxidative stress by detoxifying reactive oxygen species, including hydrogen peroxide. However, in eukaryotes, hydrogen peroxide also has important roles as a signaling molecule in the regulation of a variety of biological processes. Here, we will discuss the molecular mechanisms by which hydrogen peroxide is sensed and the increasing evidence that antioxidant enzymes play multiple, key roles as sensors and regulators of signal transduction in response to hydrogen peroxide.