The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein at a specific site with a functional group of orthogonal reactivity. Here, we achieve that goal by exploiting the intrinsic electrophilicity of the thioester intermediate formed during intein-mediated protein splicing. Detailed kinetic analyses of the reaction of nitrogen nucleophiles with a chromogenic small-molecule thioester revealed that the alpha-hydrazino acetyl group was the optimal nucleophile for attacking a thioester at neutral pH to form a stable linkage. A bifunctional reagent bearing an alpha-hydrazino acetamido and azido group was synthesized in high overall yield. This reagent was used to attack the thioester linkage between a target protein and intein, and thereby append an azido group to the target protein in a single step. The azido protein retained full biological activity. Furthermore, its azido group was available for chemical modification by Huisgen 1,3-dipolar azide-alkyne cycloaddition. Thus, the mechanism of intein-mediated protein splicing provides the means to install a useful functional group at a specific site-the C terminus-of virtually any protein.