Many α,β-unsaturated carbonyl compounds are used in biochemical and medical research. Their biological effects are due in large part to their electrophilic properties, whereby they undergo reaction with nucleophilic sites in proteins and nucleic acids. Here, we describe a structure-activity comparison of the cytotoxic properties of diethyl maleate (DEM) and closely related chemical analogs. All molecules that contained an α,β-unsaturated carbonyl group were cytotoxic to human colorectal carcinoma cells, causing apoptotic cell death. However, related molecules lacking this chemical moiety were not cytotoxic. One of the molecules screened, diethyl acetylenedicarboxylate (DAD), was considerably more cytotoxic than DEM and other analogues. Induction of cell death by DAD was significantly decreased following preincubation of cells with N-acetylcysteine, suggesting that its reactivity with thiols in cells might account for its cytotoxicity. By use of a model thiol compound, it was found that DAD can undergo addition reactions with two equivalents of thiol. When the reactivity of DAD with proteins was explored, it was determined that DAD induces oligomerization of Gpx3p, a yeast glutathione peroxidase with highly reactive cysteine residues in its active site. Our results suggest that DAD functions as a protein-thiol cross-linker, providing a potential chemical explanation for its cytotoxic potency.