Nanofiber scaffolds formed by self-assembling peptide RADA16-I have been used for the study of cell proliferation to mimic an extracellular matrix. In this study, we investigated the effect of RADA16-I on the growth of human leukemia cells in vitro and in nude mice. Self-assembly assessment showed that RADA16-I molecules have excellent self-assembling ability to form stable nanofibers. MTT assay displayed that RADA16-I has no cytotoxicity for leukemia cells and human umbilical vein endothelial cells (HUVECs) in vitro. However, RADA16-I inhibited the growth of K562 tumors in nude mice. Furthermore, we found RADA16-I inhibited vascular tube-formation by HUVECs in vitro. Our data suggested that nanofiber scaffolds formed by RADA16-I could change tumor microenvironments, and inhibit the growth of tumors. The study helps to encourage further design of self-assembling systems for cancer therapy.