We have investigated the expression of osteopontin (OPN) and CDX2 in advanced gastric cancers, and analyzed correlations with clinicopathological features to assess their prognostic potential. One-hundred and nine patients suffering from gastric cancer were recruited. Expression of OPN and CDX2 and other molecular markers was determined by immunohistochemistry. The total positive rate for OPN expression was 46.8%, with a relation to depth of cancer invasion and down regulation of intestinal markers (P<0.001), but not age, gender, or histological type. OPN was more frequently expressed in CDX2-negative (39/109=35.7%) as compared with positive lesions (12/109=11.0%) and a significant reverse correlation was noted between the two factors (P<0.001). Patients with positive OPN tumors had worse 5-year survival than those with OPN-negative cancer (P<0.001). Further analysis revealed the OPN-/CDX2+ group to have better 5-year survival than all the other three groups: OPN+/CDX2-, OPN-/CDX2- and OPN+/CDX2+. With multivariate analysis for 5-year survival, OPN was the most significant predictor of a poor prognosis of advanced gastric cancer (P=0.0043), with tumor depth of invasion as another independent indicator (P=0.0315). Osteopontin is a useful prognostic marker in gastric cancer, and combined with CDX2, may have particular advantage for predicting survival of advanced gastric cancer patients. Furthermore the present results provide a clue that in gastric cancer, CDX2 may be a transcription factor modulating the expression of osteopontin.