Objective To study the effect of knockdown activating transcription factor 3 (ATF3) on the self-renewal ability and epithelial-mesenchymal transition of colorectal cancer tumor stem cells (CRC-CSCs), and detect the changes in the secretion of immunosuppressive factors. Methods Culture the human colorectal cancer cell line HCT116, Sort CD133+ expressing CRC-CSCs cells by flow cytometry, transfect sh-ATF3 lentiviral vector to CRC-CSCs cells as the experimental group (sh-ATF3), use sh-NC lentiviral vector as a negative control group (sh-NC), normally cultured CRC-CSCs cells served as a control group, real-time fluorescent quantitative PCR (qRT-PCR) detects the relative expression of ATF3, CCK-8 method detects changes in cell activity, spheroidization test detects cell growth and renewal ability, plate clone formation test detects cell clone formation ability, immunofluorescence staining and Western Blot method were used to detect the expression of E-cadherin and Vimentin, enzyme-linked immunosorbent assay (ELISA method) detects changes in the levels of immunosuppressive factors TGF-β1, VEGF, IL-6, and IL-10 secreted by cells, western Blot detects the protein expression levels of suicide-related factors (Fas) and Fas ligand (FasL). Results Compared with the control group, the relative expression of ATF3 mRNA in CRC-CSCs cells in the sh-ATF3 group decreased (P＜0.05), and cell viability decreased at 48 h and 72 h after transfection (P＜0.05), the spheroidizing ability and the cloning ability of the cells decreased (P＜0.01), the expression of E-cadherin protein in the cells was higher and the expression of Vimentin protein was lower (P＜0.05), at the same time, the contents of TGF-β1, VEGF, and IL-10 in the cell supernatant were significantly decreased (P＜0.01), Fas protein expression increased, and FasL protein expression decreased (P＜0.05). Conclusion Knockdown of ATF3 can inhibit colorectal cancer stem cell self-renewal and epithelial-mesenchymal transition, down-regulate the expression of immunosuppressive factors TGF-β1, VEGF, IL-10, and up-regulate the expression of Fas protein and down-regulate the expression of FasL protein.