Hitherto, it has been identified that numerous basic-helix-loop-helix (bHLH) transcription factors play vital roles in tumor initiation and progression. Atonal homolog 8 (ATOH8) is a member of the bHLH family of transcription factors, which participates in embryogenesis and the development of various tissues. Several studies have demonstrated that ATOH8 is involved in the progression of malignancies; however, the effects of ATOH8 in colorectal cancer (CRC) remain unknown. The aim of the present study was to explore the expression and function of ATOH8 in CRC. The present study included 106 paired CRCs and peritumoral samples. The expression of ATOH8 was evaluated by immunohistochemistry, and the results were compared with the clinical outcomes of the patients. Furthermore, cell proliferation, cell cycle distribution, wound healing and cytotoxicity assays were performed in colon cancer cell line SW620. Immunohistochemical analyses revealed that the expression of ATOH8 in CRC tissues was significantly increased compared with the peritumoral tissues, and that the high expression of ATOH8 was associated with a high serum carcinoembryonic antigen (CEA) level and a worse overall survival. In vitro assays revealed that ATOH8 knockdown in colon cancer cells inhibited cell proliferation, induced cell cycle arrest at the S phase, and increased the percentage of apoptotic cells and sensitivity to 5-fluorouracil (5-FU). The present study suggests that ATOH8 promotes the progression of CRC and may potentially serve as a novel prognostic predictor and potential therapeutic target in CRC.