Endostatin has a compact structure with a Zn(II)-binding site (His1, His3, His11, and Asp76) at the N-terminus. In this study, the effects of Zn(II)-binding on the folding and stability of recombinant human endostatin were studied. The results show that Zn(II)-binding largely stabilizes the structure of endostatin at physiological pH. Under some proteolytic conditions, Zn(II)-binding also contributes to the integrity of the N-terminus of endostatin, which is critical for endostatin to maintain a stable structure. Moreover, engineering an extra Zn(II)-binding peptide to the N-terminus of human endostatin makes this molecule more stable and cooperative in the presence of Zn(II).