The prognosis of patients following surgery for gastric cancer is often poor and is estimated using traditional clinicopathological parameters, which can be inaccurate predictors of future survival. Kallikreins are a group of serine proteases, which are differentially expressed in many human tumors and are being investigated as potential cancer biomarkers. This study assessed the prognostic utility of human tissue kallikrein-like peptidases 6 and 10 (KLK6 and KLK10) and correlated their expression with histopathological and clinical parameters in gastric cancer. We constructed a gastric tumor tissue microarray from 113 gastrectomy specimens and quantified KLK6 and KLK10 expression using immunohistochemistry. To overcome the problem of inter-observer variability and subjectivity in immunohistochemistry interpretation, a whole-slide scanned image of the tissue microarray was analyzed using an automated algorithm to quantify staining intensity. KLK6 expression was positively correlated with nodal involvement (p=0.002) and was predictive of advanced-stage disease (p<0.05). Kaplan-Meier survival curves revealed that tumors expressing high levels of KLK6 were significantly associated with significantly lower overall survival (p=0.04). KLK10 overexpression was also a predictor of advanced-stage disease (p<0.01), but was not significantly correlated with lymph node involvement or survival period. Our results show the potential ability of KLK6 as a prognostic marker for gastric cancer.