Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both ανβ3 integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN112–136) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN112–136 inhibited binding of PTN to ανβ3 integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β3. Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing ανβ3-selective antibody. PTN112–136 did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β3 Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN112–136 may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN112–136 was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123–132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN112–136 induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to ανβ3 integrin. J. Cell. Biochem. 112: 1532–1543, 2011. © 2011 Wiley-Liss, Inc.