Colorectal cancer is the third most common type of cancer worldwide with high cell motility and metastatic potential. Reticulon 4C (RTN4-C) is the shortest isoform of the reticulon family protein RTN4, which may act to induce cell apoptosis and suppress tumor development. The aim of the present study was to determine the role of RTN4-C in colorectal cancer, and potentially identify a novel target for anti-tumor therapy. To investigate the biological role of RTN4-C in colorectal cancer, the expression levels of RTN4-C were initially analyzed in six colorectal cancer cell lines by reverse transcription-quantitative polymerase chain reaction and western blot analysis. In addition, lentivirus-based RNA interference was utilized to knock down RTN4-C expression in RKO and DLD-1 cells with low and high levels of RTN4-C, respectively. The rate of proliferation decreased in RTN4-C silenced RKO and DLD-1 cells compared with the control, as determined using MTT and colony formation assays. Flow cytometric analysis revealed that RTN4-C knockdown in RKO cells led to cell cycle arrest at the G0/G1 phase, particularly at the sub-G1 phase representing apoptotic cells. These results indicate that RTN4-C has an important role in colorectal cancer cell growth, which may provide a potential therapeutic approach for human colorectal cancer.