Abstract: N-terminal site-specific PEGylated Endostar was characterized.Peptide mapping was applied to show the occurrence of mono-PEGylation at the N-terminus of Endostar.The result demonstrated that PEG was attached to the N-terminus of Endostar.Circular dichroism spectroscopy and tryptophan emission fluorescence were used to illustrate the secondary and tertiary structure of PEGylated Endostar.Structure analysis demonstrated that PEGylation did not influence the secondary and tertiary structure of Endostar.In vitro stability experiments such as unfolding induced by urea and incubation with trypsin，as well as in vitro anti-proliferative activity in endothelial cells were performed to examine the therapeutic potential of PEGylated Endostar.PEGylated Endostar increased stability against denaturation by urea and hydrolysis by protease while retaining its anti-proliferation activity on endothelial cells.These results demonstrate that N-terminal PEGylation has improved the stability and retained the activity of Endostar in vitro.