Objective: To investigate the effects of FH535 on the self-renewal, migration and invasion of colorectal cancer stem cells (CRC-CSCs) and the mechanism. Methods: CD133+CD44+ CSCs were isolated by fluorescence-activated cell sorting (FACS) from the human DLD-1 colorectal cancer cell line. CRC-CSCs were treated with different concentrations of FH535 (20, 30, 40 μmol/L) and the half maximal inhibitory concentration (IC50) value was calculated. CRC-CSCs were treated with concentration of FH535, using untreated CRC-CSCs as control, and then, their self-renewal capacity was determined by limiting dilution assay (LDA) and sphere-forming assay, the migration and invasion abilities were measured by Transwell assay and real-time cell analysis (RTCA), and the mRNA and protein expressions of molecules involved in Wnt/β-catenin signaling pathway as well as the downstream molecules were evaluated by qRT-PCR and flow cytometry, respectively. Results: The IC50 of CRC-CSCs to FH535 was determined to be 40 μmol/L. In CRC-CSCs treated with 40 μmol/L FH535 compared with those in control group, the sphere forming capacity was significantly decreased (57.33 vs. 313.67, P<0.01) and CRC-CSCs ratio was significantly reduced (11.60/100 vs. 75.50/100, P<0.05), the migration (Transwell: 10.66 vs. 90.00; RTCA: 0.17 vs0.37) and invasion (Transwell: 8 vs. 20; RTCA: 0.14 vs. 0.37) were all significantly inhibited (all P<0.01), and the mRNA and protein expressions of LEF1 and AXIN1 in Wnt/β-catenin signaling pathway and the downstream molecules c-myc, VEGF, cyclin D1 and survivin were all significantly down-regulated (all P<0.01). Conclusion: FH535 can weaken the abilities of self-renewal, migration and invasion of CRC-CSCs, and the mechanism may be probably associated with its inhibiting the activity of Wnt/β-catenin signaling pathway.