Several different reactive oxygen species (ROS) are generated in vivo. They have roles in the development of certain human diseases whilst also performing physiological functions. ROS are counterbalanced by an antioxidant defence network, which functions to modulate ROS levels to allow their physiological roles whilst minimizing the oxidative damage they cause that can contribute to disease development. This Review describes the mechanisms of action of antioxidants synthesized in vivo, antioxidants derived from the human diet and synthetic antioxidants developed as therapeutic agents, with a focus on the gaps in our current knowledge and the approaches needed to close them. The Review also explores the reasons behind the successes and failures of antioxidants in treating or preventing human disease. Antioxidants may have special roles in the gastrointestinal tract, and many lifestyle features known to promote health (especially diet, exercise and the control of blood glucose and cholesterol levels) may be acting, at least in part, by antioxidant mechanisms. Certain reactive sulfur species may be important antioxidants but more accurate determinations of their concentrations in vivo are needed to help assess their contributions.