Aim <p>The purpose of this study is to explore the prognostic evaluation value of Ki67 and pSmad3L (S204) for gastric cancer (GC) patients by jointly analyzing their expression levels in GC tissues.</p> Methods <p>This single-center observational study enrolled 98 patients with pathologically confirmed GC, collecting 82 paired samples of tumor and adjacent normal tissues. Immunohistochemistry was utilized to detect the expression levels of CD133, E-cad, Ki67, and pSmad3L (S204). Pearson correlation coefficients were calculated to assess inter-protein relationships, while chi-square tests evaluated associations with clinicopathological parameters. Kaplan-Meier analysis generated survival curves, and univariate and multivariate COX regression analyses were conducted to establish a prognostic prediction model.</p> Results <p>Compared with adjacent normal tissues, the expressions of CD133, E-cad, and Ki67 were upregulated in GC tissues. The high expression of CD133 was associated with the tumor type, and the high expression of Ki67 was related to tumor grading and distant metastasis. Multivariate analysis showed that the depth of invasion, lymph node metastasis, Ki67, and pSmad3L (S204) were independent prognostic risk factors for GC patients. The co-expression of high levels of Ki67 and pSmad3L (S204) predicted a poor prognosis.</p> Conclusion <p>High expression of Ki67 and high expression of pSmad3L(S204) are independent risk factors for the prognosis of patients with GC, and the combined analysis of Ki67 and pSmad3L(S204) can help to improve the risk prediction.</p>