Protein S‐glutathiolation, the reversible covalent addition of glutathione to cysteine residues on target proteins, is emerging as a candidate mechanism by which both changes in the intracellular redox state and the generation of reactive oxygen and nitrogen species may be transduced into a functional response. This review will provide an introduction to the concepts of oxidative and nitrosative stress and outline the molecular mechanisms of protein regulation by oxidative and nitrosative thiol‐group modifications. Special attention will be paid to recently published work supporting a role for S‐glutathiolation in stress signalling pathways and in the adaptive cellular response to oxidative and nitrosative stress. Finally, novel insights into the molecular mechanisms of S‐glutathiolation as well as methodological problems related to the interpretation of the biological relevance of this post‐translational protein modification will be discussed.