Recent studies have demonstrated that the novel tumor suppressor protein programmed cell death 4 (PDCD4) is downregulated in several human solid cancer types and is suppressed by microRNA-21 (miR-21). The objectives of this study were: i) to establish the clinicopathological and prognostic significance of PDCD4 mRNA, and ii) to elucidate any correlation between PDCD4 mRNA and miR-21 in gastric cancer. The expression status of PDCD4 mRNA was investigated by qRT-PCR and protein expression was analyzed by an immunohistochemical study. We analyzed PDCD4 mRNA expression with respect to various clinicopathological factors in 105 gastric cancers. We also performed an association study comparing PDCD4 mRNA and miR-21 in eight cell lines and 49 gastric cancers. Expression of PDCD4 mRNA in cancer tissues was significantly lower than in non-cancer tissues (P<0.05). Patients with low PDCD4 mRNA expression was significantly correlated with size, depth, lymph node metastasis, venous invasion, advanced stage, and poor clinical prognosis (P<0.05). Expression of miR-21 in cancer tissues was significantly higher than in non-cancer tissues (P<0.05). Elevated miR-21 expression was significantly correlated with size and depth (P<0.05). An inverse correlation between PDCD4 mRNA and miR-21 was found in gastric cancer. This study revealed that low PDCD4 expression correlates with biological aggressiveness and poor prognosis in gastric cancer. Furthermore, our findings suggest that PDCD4 mRNA is negatively regulated by miR-21 in gastric cancer and may serve as a target for effective therapies.