Peroxiredoxins (Prx) are widely distributed peroxidases that can be divided into 1-Cys and 2-Cys Prx groups based on the number of conserved cysteine residues that participate in their catalytical cycle. Prx have been described to be strictly dependent on thiols, but here, we show that ascorbate (vitamin C) also reduces 1-Cys Prx, but not 2-Cys Prx, from several taxonomic groups. Reduction by ascorbate is partly related to the fact that the oxidized form of 1-Cys Prx is a stable sulfenic acid (Cys-SOH) instead of a disulfide. In addition, a histidine residue in the active site is required. In fact, we engineered a 2-Cys Prx with these two features, and it displayed ascorbate peroxidase activity. These data represent a breakthrough in the thiol-specific antioxidant paradigm. Ascorbate may be the long-sought-after biological reductant of 1-Cys Prx. Because ascorbate is present in high amounts in cells, the ascorbate/protein sulfenic acid pair represents an aspect of redox biochemistry that has yet to be explored in vivo.