Most redox-regulated chloroplast enzymes are reduced during the day and oxidized during the night. While the reduction mechanism of light-dependent enzymes is well known, the mechanism mediating their oxidation in the dark remains unknown. The thiol-dependent peroxidases, 2-Cys peroxiredoxins (Prxs), play a key role in light-dependent reduction of chloroplast enzymes. Prxs transfer reducing equivalents of thiols to hydrogen peroxide, suggesting the participation of these peroxidases in enzyme oxidation in the dark. Here, we have addressed this issue by analyzing the redox state of well-known redox-regulated chloroplast enzymes in response to darkness in Arabidopsis thaliana mutants deficient in chloroplast-localized Prxs (2-Cys Prxs A and B, Prx IIE, and Prx Q). Mutant plants lacking 2-Cys Prxs A and B, and plants overexpressing NADPH-dependent thioredoxin (Trx) reductase C showed delayed oxidation of chloroplast enzymes in the dark. In contrast, the deficiencies of Prx IIE or Prx Q exerted no effect. In vitro assays allowed the reconstitution of the pathway of reducing equivalents from reduced fructose 1,6-bisphosphatase to hydrogen peroxide mediated by Trxs and 2-Cys Prxs. Taken together, these results suggest that 2-Cys Prxs participate in the short-term oxidation of chloroplast enzymes in the dark.