Pork insulin was modified by three kinds of mPEG derivatives (mPEG-ALD, mPEG-ButyrALD and mPEG-SC) with different active moieties. The effects of reaction time, pH, molar ratio and molecular weight on the modification rate and site-selectivity were investigated. The results showed that mPEG-ButyrALD-INS had higher site-selectivity and modification rate compared to the other two modifiers characterized by HPLC, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and trinitrobenzene sulfonic acid (TNBS). The secondary structure of the mPEG-ylated insulins was studied by circular Dichroism (CD). It is found that the secondary structure of insulin was changed hardly after mPEGylation. The in vivo experiment results showed that mPEG-ButyrALD-INS displayed longer half-life and extended action profiles.