Objective To investigate the effect and mechanism of B7-H4 expression on the prognosis of patients with gastric cancer. Methods The databases of Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN and Kaplan-Meier Plotter were used to analyze the expression of B7-H4 in gastric cancer tissues and its relationship with the prognosis of gastric cancer patients. Immunohistochemistry staining was performed to observe the expression of B7-H4 protein in gastric cancer tissues and analyze its correlation with clinicopathological characteristics, cell cycle regulatory proteins, microvessel density (MVD), stemness-related proteins, and PI3K/Akt/NF-κB signaling pathway proteins. Immunofluorescence and Western blotting were used to detect the expressions of B7-H4 and stemness-related proteins in gastric cancer cells. Oncomine database was employed to analyze the correlation between B7-H4 and the expressions of PI3K and Akt mRNA in gastric cancer tissues. Results The expression level of B7-H4 was higher in gastric cancer tissues than in normal tissues, and its expression was significantly related to tumor differentiation, pT staging, lymph node metastasis, distant metastasis and clinical staging (P<0.05), and indicated a poor prognosis in gastric cancer. The expression of B7-H4 was positively correlated with the expressions of cell cycle regulatory protein p16 (r=0.250, P<0.001) and stemness-related proteins LSD1 (r=0.331, P<0.001), SOX9 (r=0.176, P=0.018) and CD133 (r=0.434, P<0.001). The MVD was significantly higher in B7-H4 positive tissue than in B7-H4 negative tissue (P=0.019). Furthermore, B7-H4 was co-expressed with stemness-related proteins LSD1, SOX9, and CD133 in gastric cancer cell lines, and the expression of B7-H4 was positively correlated with the expressions of p-PI3K-p85 (r=0.269, P<0.001), p-Akt-Ser473 (r=0.282, P<0.01), p-Akt-Thr308 (r=0.214, P=0.005) and NF-κB p65 (r=0.157, P=0.034). The expression of B7-H4 in gastric cancer tissue was positively correlated with the expressions of PI3K mRNA (r=0.403, P<0.001) and Akt mRNA (r=0.351, P<0.001). Conclusion B7-H4 can promote gastric cancer progression and is closely related to poor prognosis, and the mechanism may be related to the regulation of gastric cancer stemness via PI3K/Akt/NF-κB.