Deregulated PIN1 is associated with cancer development and progression. Herein, for the first time, we evaluate the roles that PIN1 in tumorigenic characteristics of colorectal cancer (CRC) cells.In this study, PIN1 expression was knocked down in SW-48 cells by synthetic small interfering RNA (siRNA). After confirming the knockdown of PIN1, cell viability, colony formation, apoptosis, autophagy, cancer stem cell (CSC)-related genes, CSC-related signaling pathways, cell migration, and 5-FU chemosensitivity were evaluated in vitro.Transfection of PIN1 siRNA into SW-48 cells inhibited cancer cell proliferation, migration, and increased apoptosis and autophagy. Transfected SW-48 cells had lower properties of CSCs through the inhibition of β-catenin and Notch1 gene expression. Moreover, inhibition of PIN1 enhanced the inhibitory effect of 5-FU on SW-48 cell proliferation.Our results indicated that targeting of PIN1 serves as a promising therapeutic solution for the suppression of tumor progression processes in CRC.