Aberrant activation of Wnt signaling plays a critical role in the initiation and progression of colorectal cancer (CRC). Chlorquinaldol (CQD) is a topical antimicrobial agent used to treat skin infections. Little is known about the anticancer activity of CQD and its underlying mechanisms. In this study, CQD was demonstrated to inhibit Wnt/β-catenin signaling through targeting the downstream part of this pathway. The results showed that CQD could inhibit the acetylation of β-catenin and disrupt the interaction of β-catenin with T-cell factor 4 (TCF4), leading to reduced binding of β-catenin to the promoters of Wnt target genes and downregulation of the expression of these target genes. Moreover, treatment with CQD suppressed the proliferation, migration, invasion and stemness of CRC cells. In APC mice and CRC cell xenografts, administration of CQD suppressed tumor growth and the expression of Wnt target genes c-Myc and Leucine-rich G protein-coupled receptor-5 (LGR5). These results strongly suggest that CQD may be a promising therapeutic agent in the treatment of CRC. CRC, colorectal cancer; CSC, cancer stem cell; TCF4, T-cell factor 4; LGR5, Leucine-rich G protein-coupled receptor-5; APC, adenomatous polyposis coli; LEF, lymphoid enhancing factor; ZNRF3, zinc and ring finger 3; RNF43, ring finger protein 43; RSPO, R-spondin; CtBP, C-terminal-binding protein; GRO/TLE, groucho/transducin-like enhancer of split; HDAC, histone deacetylase; CBP, CREB-binding protein; PCAF, p300/CBP-associated factor; CQD, Chlorquinaldol; 8-HQ, 8-hydroxyquinoline; DMEM, Dulbecco’s Modified Eagle’s Media; IMDM, Iscove’s Modified Dulbecco’s Medium; NFAT, nuclear factor of activated T cells; SOX2, sex determining region Y-box 2; ChIP, chromatin immunoprecipitation; BCA, bicinchoninic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, poly-vinylidenefluoride; LiCl, lithium chloride; PBT, Panobinostat; TNIK, Traf2- and Nck-interacting kinase.Copyright © 2020. Published by Elsevier Ltd.