Increased expression of polypyrimidine tract-binding protein 1 (PTBP1) has been observed in human ovarian tumors, glioblastomas, and breast cancer, but its biological roles in tumorigenesis is not fully clear. In the present research, we investigated the biological role of PTBP1 in colon cancer. We found that PTBP1 was overexpressed both in colon cancer cell lines and tissues. Tissue microarray analysis (TMA) indicated that low PTBP1 expression predicted a favorable overall survival for colon cancer patients. Using small interfering RNA technology, we found that down-regulation of PTBP1 significantly inhibited colon cancer cell growth/proliferation, and induced cell cycle arrest as well as apoptosis in vitro. Western blot analysis showed that siRNA PTBP1 could up-regulate the expression of cytoC, p53 and Bax as well as down-regulated p85, p-AKT, cyclinD1, CDK4 and Bcl2 compared to the control. Furthermore, Caspase-3 and PARP1 were activated when PTBP1 is knockdown. This study implies that PTBP1 plays an important role in tumorigenesis of colon cancer.