Noncovalent interactions are ubiquitous in biology, taking on roles that include stabilizing the conformation of and assembling biomolecules, and providing an optimal environment for enzymatic catalysis. Here, we describe a noncovalent interaction that engages the sulfur atoms of cysteine residues and disulfide bonds in proteins-their donation of electron density into an antibonding orbital of proximal amide carbonyl groups. This n→ π* interaction tunes the reactivity of the CXXC motif, which is the critical feature of thioredoxin and other enzymes involved in redox homeostasis. In particular, an n→ π* interaction lowers the p Ka value of the N-terminal cysteine residue of the motif, which is the nucleophile that initiates catalysis. In addition, the interplay between disulfide n→ π* interactions and C5 hydrogen bonds leads to hyperstable β-strands. Finally, n→ π* interactions stabilize vicinal disulfide bonds, which are naturally diverse in function. These previously unappreciated n→ π* interactions are strong and underlie the ability of cysteine residues and disulfide bonds to engage in the structure and function of proteins.