Benzoylformate decarboxylase (BFD) from Pseudomonas putida is a thiamin diphosphate-dependent enzyme that catalyzes the non-oxidative decarboxylation of benzoylformate. Here we report the discovery of a mechanism-based inhibitor of BFD that is unusual in that it covalently modifies the enzyme via active site phosphorylation. Incubation of BFD with benzoylphosphonate results in time- and concentration-dependent inactivation of the enzyme. X-ray crystallography reveals that the inactivation is due to the phosphorylation of an active site serine residue.