Glutaredoxins (GRXs) are small ubiquitous disulfide oxidoreductases known to use reduced glutathione (GSH) as electron donor. In photosynthetic organisms, little is known about the biochemical properties of GRXs despite the existence of around 30 different isoforms in higher plants. We report here the biochemical characterization of Chlamydomonas GRX1 and GRX3, the major cytosolic and chloroplastic isoforms, respectively. Glutaredoxins are classified on the basis of the amino acid sequence of the active site. GRX1 is a typical CPYC-type GRX which is reduced by GSH and exhibits disulfide reductase, dehydroascorbate reductase and deglutathionylation activities. In contrast, GRX3 exhibits unique properties. This chloroplastic CGFS-type GRX is not reduced by GSH and has an atypically low redox potential (-323 ± 4 mV at pH 7.9). Remarkably, GRX3 can be reduced in the light by photoreduced ferredoxin and ferredoxin-thioredoxin reductase. Both GRX proved to be very efficient catalysts of A4-glyceraldehyde-3-phosphate dehydrogenase (A4-GAPDH) deglutathionylation, while cytosolic and chloroplastic thioredoxins were inefficient. Glutathionylated A4-GAPDH is the first physiological substrate identified for a CGFS-type GRX.