Osteopontin (OPN) plays an important role in tumorigenesis, tumor invasion, and metastasis in many types of cancers, including gastric cancer. Recently, much interest has been focused on the role of OPN in tumor angiogenesis. Our previous studies have shown that OPN is overexpressed, and associated with mean microvessel density in, the tissue samples of patients with gastric cancer. In the present study, we aimed to further determine and provide evidence for the role of OPN in gastric-cancer-associated angiogenesis by diminishing OPN expression in gastric cancer cells using the small interference RNA method, and then evaluate the effects of OPN on gastric cancer-associated angiogenesis by in vivo and in vitro assays. Our results revealed that reduced OPN production by gastric cancer cells would reduce the proliferation, migration, and tube formation of human umbilical vein endothelial cells, and lead to a lower microvessel density, i.e., angiogenesis, in transplanted tumors of mice. These data confirm the positive role of OPN in gastric-cancer-associated angiogenesis.