To elucidate the biological functions of osteopontin (OPN) in gastric tumors and to better understand the molecular events of OPN responsible for the malignancy, the present studies were performed. Growth curve, apoptosis assay, invasion assay and migration assay revealed that OPN status significantly affected proliferation, apoptosis, invasion and migration in gastric cancer cell lines. In mouse xenograft models of human gastric cancer, OPN silencing significantly inhibited tumor growth and the incidence of metastasis compared with non-silenced control. Mechanistic investigations revealed that OPN silencing inhibited the mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and phosphoinositide 3-kinase (NF-κB) pathways, and OPN-mediated NF-κB activity was reduced in the presence of MAPK or PI3K inhibitor. Our findings also indicated that OPN through the NF-κB pathway promotes the expression of matrix metalloproteinase 2 (MMP-2), MMP-9 and urokinase-type plasminogen activator (uPA), the activation of MMP-2 and MMP-9, and the inhibition of caspase-3. Taken together, our results reveal that OPN promotes the progression of gastric cancer through the NF-κB pathway, which is regulated by the MAPK and PI3K pathways and leads to MMP-2, MMP-9 and uPA activation and caspase-3 inhibition. These findings identify OPN as a novel oncogene in gastric cancer and suggest that OPN is an attractive therapeutic target for this aggressive malignancy.