The substance P (SP)/neurokinin-1 receptor (NK1R) complex and the Wnt cascade are pivotal signaling pathways in the regulation of cell growth and hence, potent targets for future anticancer therapies. However, while the Wnt cascade has long been associated with colon cancer, little is known about the expression of the NK1R complex as a potential target in this tumor and its molecular basis in tumorigenesis in general. We treated the human colon cancer cell lines LiM6 and DLD1 with the NK1R antagonist and the clinical drug aprepitant (AP) and analyzed both growth response and downstream mechanisms using MTT-assay, reverse phase protein array (RPPA), western blot, Super TOP/FOP, confocal microscopy, and sphere formation ability (SFA) assays. Following NK1R blockage, we found significant growth inhibition of both colon cancer cell lines. When analyzing downstream mechanisms, we found a striking inhibition of the canonical Wnt pathway represented by decreased Super TOP/FOP and increased membrane stabilization of β-catenin. This effect was independent from baseline Wnt activity and mutational status of β-catenin. Further, treatment of colon cancer cells grown under cancer stem cell (CSC) conditions reduced sphere formation in both number and size after a single treatment period. We show that the NK1R can be a potent anticancer target in colon cancer and that NK1R antagonists could potentially serve as future anticancer drugs. This effect was seen not only in primary cancer cells but, for the first time, also in CSC-like cells, potentially including these cells in a therapeutic effect. Also, we describe the robust inhibition of canonical Wnt signaling through targeting the SP/NK1R signaling cascade. These findings give important insight into the molecular mechanisms of the SP/NK1R complex as a critical component in tumorigenesis and could help to identify future anticancer therapies for colon and other Wnt-activated cancers.