For site-selective chemical modification in the vicinity of the active site of chymotrypsin (Csin), a peptide derivative 1 bearing a diphenyl 1-amino-2-phenylethylphosphonate moiety for targeting the active site and an active ester moiety for anchoring to lysine residues was designed and synthesized. Most of Csin was inactivated by 1 for 10 mm and then reactivated by incubation with 2-pyridinaldoxime methiodide to give modified Csin. Mass spectrometric analysis of modified Csin showed that 1 was selectively anchored at Lys175.