KLK6 encoding kallikrein-related peptidase 6, a trypsin-like serine protease, has been shown to be upregulated in several cancers, although the tumorigenic role of KLK6 has not been elucidated. In this study, KLK6 was identified as a highly upregulated gene in gastric cancer; therefore, the possibility that KLK6 might be a suitable candidate tumor marker was examined. RT-PCR and immunohistochemical analysis showed overexpression of KLK6 in gastric cancer tissues compared to nontumor regions. Sera from gastric cancer patients had a 1.7-fold increase in KLK6 (373.1 μg/L, P = 0.048) compared to healthy individuals (214.2 μg/L), although there was no significant difference among patients with various tumor stages. Cellular invasiveness decreased by 45% in cells transfected with KLK6-specific small interfering RNA. Exogenous overexpression of KLK6 led to decreased activity of the E-cadherin promoter. This study shows that KLK6 is significantly upregulated and secreted in gastric cancer tissues and sera, suggesting that KLK6 might be used as a potential biomarker and therapeutic target for gastric cancer.