T lymphocyte-mediated immune responses are critical for antitumour immunity; however, T cell function is impaired in the tumour environment. MicroRNAs are involved in regulation of the immune system. While little is known about the function of intrinsic microRNAs in CD8(+) T cells in the tumour microenvironment. Here, we found that miR-491 was upregulated in CD8(+) T cells from mice with colorectal cancer. Retroviral overexpression of miR-491 in CD8(+) and CD4(+) T cells inhibited cell proliferation and promoted cell apoptosis and decreased the production of interferon-γ in CD8(+) T cells. We found that miR-491 directly targeted cyclin-dependent kinase 4, the transcription factor T cell factor 1 and the anti-apoptotic protein B-cell lymphoma 2-like 1 in CD8(+) T cells. Furthermore, tumour-derived TGF-β induced miR-491 expression in CD8(+) T cells. Taken together, our results suggest that miR-491 can act as a negative regulator of T lymphocytes, especially CD8(+) T cells, in the tumour environment; thus, this study provides a novel insight on dysfunctional CD8(+) T cells during tumourigenesis and cancer progression. In conclusion, miR-491 may be a new target for antitumour immunotherapy.