Previous studies have demonstrated the cancer-type specific role of forkhead box protein A1 (FOXA1) in human malignancies. However, the clinical significance of FOXA1 and its biological function in gastric cancer remain unknown. In this study, the expression of FOXA1 in 80 pairs of gastric cancer tissues and corresponding non-tumor tissues was analyzed using immunohistochemistry and quantitative real-time polymerase chain reaction. We found that the levels of FOXA1 protein and mRNA in gastric cancer tissues were significantly higher than those in matched tumor-adjacent tissues. Furthermore, clinical association analysis indicated that the positive expression of FOXA1 was associated with adverse clinicopathological characteristics of gastric cancer patients including poor tumor differentiation, large tumor size, and advanced tumor-node-metastasis tumor stage. Notably, gastric cancer patients with positive expression of FOXA1 had a poorer 5-year overall survival and recurrence-free survival. In addition, FOXA1 knockdown remarkably inhibited cell proliferation and induced apoptosis in both SGC-7901 and MGC-803 cells. In vivo studies indicated that FOXA1 knockdown prominently suppressed tumor growth of gastric cancer in a nude mouse xenograft model. Mechanistically, we disclosed that the expression of Yes-associated protein was decreased accordingly after FOXA1 knockdown in both SGC-7901 and MGC-803 cells. Taken together, our data suggest that FOXA1 may serve as a promising prognostic indicator and an attractive therapeutic target of gastric cancer.