Anticancer bioactive peptide (ACBP), a novel bioactive peptide isolated from spleens of goats immunized with tumor extracts in our lab, can inhibit the proliferation of CRC in vitro and vivo. However, it remains unclear how the proliferation of CRC is inhibited by ACBP at the molecular level. Here, we provide evidences showing that ACBP significantly inhibits the expression of Wnt/β-catenin related genes (cyclin D1, met and c-myc) through pharmacotranscriptomic and qRT-PCR analysis in CRCs. Active β-catenin, a key protein within Wnt pathway, was compromised remarkably by ACBP in three CRCs, including HCT116, RKO and HT29. Thus nuclear accumulation of active β-catenin was retarded and finally lead to the decreased expression of oncogenes cyclin D1, met, and c-myc. In addition, we proved that active β-catenin reduction was mainly due to the inhibition of phospho-LRP6 and stimulation of phospho-β-catenin by ACBP. Based on the detection of Met and C-Myc in CRC tumor tissue without prior radiotherapy or chemotherapy, our results demonstrated that ACBP can act as a promising anticancer agent for CRC by targeting Wnt/β-catenin pathway, especially active β-catenin.