proEMAPⅡ/p43 was originally described as a scaffolding protein that is a component of the multi-aminoacyl-tRNA synthetase complex. Recently, proEMAPⅡ/p43 was found to be a cytokine as well as an endogenic anti-angiogenic protein. The p43 protein is thought to be a precursor of endothelial monocyte-activating polypeptideⅡ (EMAPⅡ). p43 showed higher biological activity than EMAPⅡ, making it a promising anti-angiogenesis inhibitors for cancer therapy. However, the structure of p43 and its function in angiogenesis remain unknown. Here, we constructed p43-like proteins with low molecular mass and high activity. We also determined the functional domains for the anti-angiogenic activity of p43. First, we predicted the secondary structure of p43 using a bioinformatics method, and then constructed 10 p43 truncated mutants. We compared the anti-angiogenic activity of the full-length p43 with the activities of the truncated proteins. We found that all the truncated proteins inhibited the migration of endothelial cells and prevent tubule formation. The deletion of up to 79 amino acids at the N-terminus or 47 amino acids at the C-terminus of p43 increased the activity to 2～3 times that of full-length p43. We identified three p43 truncations with lower molecular mass and higher activity than the full-length p43. These findings will help improve our understanding of the structure and function of p43, which, in turn, will be helpful to the further studies on the possible clinical applications of p43-like drugs.