Persulfides (RSSH/RSS2) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS2), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH.The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS2 presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called a effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the a effect correlated with the Brønsted nucleophilic factor, bnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of thea effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.