The members of the kallikrein-related peptidase (KLK) family are aberrantly expressed in cancer, including colorectal adenocarcinoma. KLK4 is an endogenous activator of protease-activated receptor 1 (PAR1) in HT-29 colorectal adenocarcinoma cells, inducing PAR1 signaling and subsequent ERK1/2 activation. The aim of this study was to analyze KLK4 mRNA expression in colorectal adenocarcinoma and to examine its prognostic value as a novel molecular tissue biomarker in this malignancy. Therefore, total RNA was isolated from primary tumors of 81 colorectal adenocarcinoma patients, cDNA was prepared, and KLK4 mRNA expression analysis was performed using quantitative real-time PCR. KLK4 mRNA was significantly associated with the Dukes stage, tumor invasion, size, and histological grade. Survival analysis demonstrated that KLK4 mRNA expression constitutes an unfavorable prognostic biomarker in colorectal adenocarcinoma, predicting poor disease-free survival (DFS), independently of the nodal status and tumor size. Furthermore, KLK4 mRNA predicts short-term relapse of lymph node-negative patients or those with tumors of early Dukes stage. In conclusion, KLK4 mRNA expression can be regarded as a novel potential tissue biomarker in colorectal adenocarcinoma.