〔Abstract〕 Objective To study the expression levels of miR-486-5 p in colorectal cancer(CRC) cell lines and CRC stem cells(CSCs), and to explore its effect on CRC CSCs stemess and the targeted regulation of miR-486-5 p on Forkhead box class O1(FOXO1). Methods qRT-PCR was used to detect the expression levels of miR-486-5 p in CRC cell lines SW480, HCT116, SW620 and HT29 and normal colon cell line NCM460. CSCs were cultured in HCT116 cells. Western blot was used to detect the expressions of Nanog, SOX2, OCT4 stemess marker protein and miR-486-5 p in CSCs. After transfected with miR-486-5 p mimic and control(NC) 48 h, MTS was used to detect the proliferation ability of CSCs, Transwell assay was used to detect the metastasis ability of CSCs, and Boyden assay was used to detect the invasive ability of CSCs. CSCs stably overexpressing miR-486-5 p were screened by geneticin(G418) and injected into BALB/c nude mice to observe the effect of miR-486-5 p on the tumorigenic ability of CSCsin vivo. The targeting effect of mir-486-5 p on FoxO1 gene was verified by targetscan 7.1 software prediction and double luciferase reporter gene test. qRT-PCR was used to detect the effect of miR-486-5 p on FOXO1 mRNA expression in CSCs. Results qRT-PCR results showed that the expression of miR-486-5 p in CRC cell lines was lower than that in normal colon cell line(P<0.05). The expressions of OCT4, Nanog and SOX2 stem cell markers were up-regulated in CSCs, and the expression of miR-486-5 p decreased in CSCs. The proliferation, metastasis and invasion of CSCs decreased after miR-486-5 p mimic transfected(P<0.05). The tumorigenic ability of CSCsmiR-486-5 pnude mice decreased(P<0.05). TargetScan7.1 prediction software and double luciferase reporter gene assay confirmed that FOXO1 was a target gene of miR-486-5 p, and the expression of FOXO1 mRNA in miR-486-5 p mimic group decreased(P<0.05). Conclusion miR-486-5 p has low expression in CRC cell lines and CSCs, and miR-486-5 p may inhibit the stemness of CSCs by negative regulation of FOXO1.