Osteopontin (OPN) is a phosphorylated glycoprotein secreted into body fluids by various cell types. OPN contains arginine-glycine-aspartate (RGD) and serine-leucine-alanine-tyrosine (SLAY) motifs that bind to several integrins and mediate a wide range of cellular processes. In the present study, the proangiogenic effects of a 20-amino-acid OPN peptide (OPNpt20) containing RGD and SLAY motifs were examined in human umbilical vein endothelial cells (HUVECs) and in a rat focal cerebral ischemia model. OPNpt20 exerted robust proangiogenic effects in HUVECs by promoting proliferation, migration and tube formation. These effects were significantly reduced in OPNpt20-RAA (RGD->RAA)-treated cells, but only slightly reduced in OPNpt20-SLAA (SLAY->SLAA)-treated cells. Interestingly, a mutant peptide without both motifs failed to induce these proangiogenic processes, indicating that the RGD motif is crucial and that SLAY also has a role. In OPNpt20-treated HUVEC cultures, AKT and ERK signaling pathways were activated, but activation of these pathways and tube formation were suppressed by anti-αvβ3 antibody, indicating that OPNpt20 stimulates angiogenesis via the αvβ3-integrin/AKT and ERK pathways. The proangiogenic function of OPNpt20 was further confirmed in a rat middle cerebral artery occlusion model. Total vessel length and vessel densities were markedly greater in OPNpt20-treated ischemic brains, accompanied by induction of proangiogenic markers. Together, these results demonstrate that the 20-amino-acid OPN peptide containing RGD and SLAY motifs exerts proangiogenic effects, wherein both motifs have important roles, and these effects appear to contribute to the neuroprotective effects of this peptide in the postischemic brain.