Programmed cell death 6 (PDCD6) plays an important role in apoptotic cell death and tumorigenesis. In this study, we investigated whether PDCD6 contributes to the development and/or progression of gastric cancers. PDCD6 protein expression was examined in 169 advanced gastric cancer specimens by immunohistochemistry and then correlated with clinicopathologic parameters. We also analyzed mutations, methylation status, and alterations in DNA copy number and mRNA transcripts, and protein expression of PDCD6 in gastric cancers. The effect of PDCD6 on cell viability and death was further examined in wild- and mutant-type PDCD6 transfected AGS and HEK293T cell lines. Increased expression of PDCD6 expression was detected in 124 (73.4%) out of 169 gastric cancer specimens. Statistically, altered expression of PDCD6 was closely associated with survival rates (P = 0.0069). One non-sense mutation was found at codon 175 of PDCD6, and no hypermethylation was found in gastric cancers. Decreased copy numbers and mRNA expression of PDCD6 were found in 7 (16.7%) and 10 (23.8%) of 42 gastric cancer specimens, respectively. AGS and HEK293T cells transfected with wild-type PDCD6 showed marked inhibition of cell viability and induction of cell death via activation of mitochondrial cell death pathways, whereas mutant-type PDCD6 showed partial ablation of tumor suppressor activity. In addition, AGS cells transfected with wild-type PDCD6 and treated with 5-FU showed synergistic inhibition of cell viability (P < 0.001). These data provide evidence that the PDCD6 gene is a significant prognostic biomarker for advanced gastric cancer patients.