Among several chemotherapeutic agents, 5-fluorouracil (5-FU) has been widely used as a key drug in adjuvant chemotherapy for gastric cancer. However, no reliable marker, which predicts the response to 5-FU in an adjuvant setting, has been identified. Hypoxia-induced drug resistance, via upregulation of HIF-1alpha, is a major obstacle in the development of effective cancer therapy. However, few clinical studies have so far assessed the relationship between the HIF-1alpha expression and the chemo-resistance of gastric cancer patients in an adjuvant setting. We established 2 HIF-1alpha knockdown gastric cancer cell lines in order to clarify the role of HIF-1alpha in chemo-resistance against 5-FU. Furthermore, expression of HIF-1alpha was immunohistochemically assessed in 91 resected specimens. Sixty-four of 91 patients received 5-FU adjuvant chemotherapy after surgery. HIF-1alpha expression was associated with the significantly shorter relapse-free survival and disease-specific survival in the 64 patients of adjuvant group (p = 0.026, 0.014, respectively), but not in the 27 of surgery group. Multivariate analysis showed that HIF-1alpha was an independent risk factor for relapse in 64 patients in the adjuvant group (p = 0.029). In conclusion, the current study confirmed, for the first time that HIF-1alpha expression is an independent risk factor for relapse in high-risk gastric cancer patients who underwent curative surgery followed by adjuvant 5-FU chemotherapy. A favorable effect of 5-FU might therefore be expected in patients that do not express HIF-1alpha, whereas, other types of chemotherapy or additional treatments, such as HIF-1alpha inhibitors, should be considered in patients that do express HIF-1alpha.