The purpose of this study was to evaluate expression and prognostic impact of Nanog, Oct4, Sox2, proliferation cell nuclear antigen (PCNA), Ki67 and E-cadherin in patients with gastric cancer (GC) by immunohistochemistry. A total of 69 patients were recruited who underwent gastrectomy between 2008 and 2009. We found that expression levels of Nanog, Oct4, Sox2, PCNA, Ki67 and E-cadherin were 26.1, 53.6, 49.3, 52.2, 60.9 and 60.9 %, respectively. Co-expression of more than any two proteins (defined as high-risk group) was detected in 43 of 69 (62.3 %) patients with GC. Only positive expression of Oct4 had relationship with lymphatic invasion (p = 0.013), and positive expression of Ki67 was correlated with T classification (p = 0.011). Furthermore, positive expression of Oct4 (p = 0.043), PCNA (p = 0.035) and Ki67 (p = 0.023) was significantly associated with poor 3-year disease-free survival (DFS). The same result was detected in patients with E-cadherin reduced expression (p = 0.022). But only PCNA positive expression predicted poor overall survival (p = 0.042) in univariate analysis. In addition, 3-year DFS was 20 % in high-risk group and 71 % in low-risk group. The same tendency was found between OS and co-expression of proteins. There was a remarkable difference between DFS or OS and co-expression of more than two proteins (p = 0.000). Multivariate analysis showed that E-cadherin and co-expression were independent prognostic factors of 3-year diseases-free survival. But only co-expression of more than two markers dramatically affected the survival of GC patients. These findings provide evidence that combined evaluation of Nanog, Oct4, Sox2, PCNA, Ki67 and E-cadherin may be a more powerful prognostic factor to predict relapse and distant metastasis for patients with GC.