S-Glutathionyl-hydroquinone reductases (GS-HQRs) are a new class of glutathione transferases, widely present in bacteria, halobacteria, fungi, and plants. They catalyze glutathione (GSH)-dependent reduction of GS-trichloro-p-hydroquinone to trichloro-p-hydroquinone. Since GS-trichloro-p-hydroquinone is uncommon in nature, the extensive presence of GS-HQRs suggests they use common GS-hydroquinones. Here we demonstrate that several benzoquinones spontaneously reacted with GSH to form GS-hydroquinones via Michael addition, and four GS-HQRs from yeast and bacteria reduced the GS-hydroquinones to the corresponding hydroquinones. The spontaneous and enzymatic reactions led to the reduction of benzoquinones to hydroquinones with the concomitant oxidation of GSH to oxidized glutathione (GS-SG). The enzymes did not use GS-benzoquinones or other thiol-hydroquinones, for example, S-cysteinyl-hydroquinone, as substrates. Apparent kinetic parameters showed the enzymes preferred hydrophobic, bulky substrates, such as GS-menadiol. The broad substrate range and their wide distribution suggest two potential physiological roles: channeling GS-hydroquinones back to hydroquinones and reducing benzoquinones via spontaneous formation of GS-hydroquinones and then enzymatic reduction to hydroquinones. The functions are likely important in metabolic pathways with quinone intermediates.