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The purpose of this study was to optimize an Exendin-4 (Ex4-Cys) site-specific PEGylation method with a high-molecular weight trimeric PEG.Here, we describe the preparation of C-terminal specific PEGylated Ex4-Cys (C40-tPEG-Ex4-Cys), which was performed using cysteine and amine residue specific coupling reactions using Ex4-Cys and activated trimeric PEG.The C40-PEG-Ex4-Cys obtained at high yields (~ 83 %) and characterized by MALDI-TOF mass spectrometry.The receptor binding affinity of C40-PEG5K-Ex4-Cys was 3.5-fold higher than that of N-terminal PEGylated Ex4-Cys (Nter-PEG5K-Ex4-Cys), and receptor binding by the trimeric PEG (tPEG;23, 50 kDa) adduct was much higher than that of branched PEG (20 kDa).Furthermore, C40-tPEG50K-Ex4-Cys was found to have greater blood circulating t1/2 and AUCinf values than native Ex4-Cys by 7.53 and 45.61-fold, respectively.Accordingly, its hypoglycemic duration was much greater than that of native Ex4-Cys at 59.2 h, at a dose of 25 nM/kg (native Ex4-Cys 7.3 h).The results of this study show that C-terminal specific PEGylation using trimeric PEG is effective when applied to Ex4-Cys, and suggest that C40-tPEG50K-Ex4-Cys has considerable potential as a type 2 anti-diabetic agent.