Regenerating gene family members 1 (REG Ialpha) and 4 (REG IV) are overexpressed in a subset of gastric cancers. However, comparative characterization of the expression of these family proteins has remained unclear. Therefore, we aimed to elucidate not only the association between REG protein expression and mucin phenotype but also their significance as a prognostic marker for patients with gastric cancer. The expression of REG Ialpha, REG IV, CDX2, MUC2, and MUC5AC in gastric cancer tissues was examined by immunohistochemistry. The relationship between REG protein expression and clinicopathological parameters or mucin phenotype was then analyzed. REG Ialpha and REG IV expression was positive in 33 (52%) and 31 (49%) of 63 gastric cancers examined, respectively. REG Ialpha expression was significantly related to venous invasion and tumor stage, whereas REG IV expression showed no relationship to clinicopathological features. With regard to mucin phenotype, REG IV expression was significantly correlated with MUC2 and CDX2 expression, suggesting an association with the intestinal mucin phenotype of gastric cancer. On the other hand, REG Ialpha expression had no correlation with MUC2, CDX2, or MUC5AC in gastric cancer tissues. Expression of REG Ialpha but not REG IV was an independent predictor of poor outcome in patients with gastric cancer. In addition, patients with gastric cancer negative for both REG Ialpha and REG IV expression had a significantly better outcome than patients positive for either REG Ialpha or REG IV. Profiling of REG protein expression is useful to for prognostication of patients with gastric cancer.