Autophagy plays a key role in colorectal cancer (CRC) development and reduces the sensitivity of CRC cells to treatment. The present study reported a novel tumor-suppressive role for autophagy, which was demonstrated to be regulated through the novel oncogene neurotrophin-4 (NTF4). NTF4 was significantly overexpressed in tumor tissue compared with non-tumor mucosa, and the upregulation of NTF4 in CRC was associated with poor overall survival and advanced TNM stage. The genetic knockdown of NTF4 using short hairpin RNA in CRC cells prevented epithelial-to-mesenchymal transition and activated autophagy; this was regulated through the interaction between autophagy-associated gene 5 (Atg5) and the mitogen-activated protein kinase pathway. In addition, the knockdown of NTF4 inhibited cell invasion, migration, proliferation and colony formation, and promoted cell cycle arrest. Treatment of the cells with the autophagy inhibitor chloroquine (CQ) rescued these functions and promoted cell invasion, migration, proliferation and colony formation. Finally, the knockdown of NTF4 inhibited the growth of subcutaneous xenografts in Balb/c-nu mice. In conclusion, these findings suggested that NTF4 may be a diagnostic marker associated with the overall survival and progression of patients with CRC. NTF4 was found to promote tumorigenesis and CRC development through autophagy regulation.