Aberrant expression of pancreatic adenocarcinoma upregulated factor (PAUF), a novel secretory protein, has been reported in several types of cancer. However, in colorectal cancer (CRC), whether PAUF also plays its oncogenic role through the Wnt/β-catenin pathway and its effect in regulating malignant phenotypes of CRC is unknown. In this study, we detected PAUF and β-catenin expression levels by immunohistochemical analysis and real-time PCR in CRC tissues, adjacent non-tumor tissues (NATs) and 5 CRC cell lines. The results demonstrated that the expression of PAUF and β-catenin in tumor tissues was higher than in NATs. Moreover, the expression of PAUF was correlated with the expression of β-catenin in both tumor tissues and NATs. The HCT116 cell line, which has the highest PAUF expression of the 5 cell lines, was transfected with small interfering RNA (siRNA) targeting on PAUF, which significantly downregulated the expression of PAUF in cancer cells. Successful transfection was confirmed by using RT-PCR and western blot analysis. Further studies demonstrated that PAUF-siRNA inhibited the proliferation of CRC cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. At the same time, PAUF-siRNA inhibited the invasion, adhesion and migration of the tumor cells. In conclusion, this study suggested that PAUF was expressed in CRC at a high frequency. Interference of PAUF may be an effective strategy for regulating malignant phenotypes of CRC through the Wnt/β-catenin pathway.