Cancer stemness drives tumor progression and drug resistance, representing a challenge to cancer eradication. Compelling evidence indicates that cancer cells can reenter the stem cell state due to the reprogramming of self-renewal machinery. Here, we show that CD146 knockdown induces stem cell properties in colorectal cancer (CRC) cells through activating canonical Wnt signaling. shRNA-mediated CD146 knockdown in CRC cells facilitates tumor initiation in serial xenotransplantation experiments. Moreover, upon CD146 knockdown, CRC cells show elevated expression of specific cancer stem cell (CSC) markers, increased sphere and clone formation as well as drug resistance in vitro. Mechanistically, our findings provide evidence that CD146 expression negatively correlates with canonical Wnt/beta-catenin activity in CRC cell lines and primary CRC specimens. Knockdown of CD146 results in inhibition of NF-kappa B/p65-initiated GSK-3 beta expression, subsequently promoting nuclear translocation and activation of beta-catenin, and as a consequence restoring stem cell phenotypes in differentiated CRC cells. Together, our data strongly suggest that CD146 functions as a suppressor of tumorigenesis and cancer stemness in CRC through inactivating the canonical Wnt/beta-catenin cascade. Our findings provide important insights into stem cell plasticity and the multifunctional role of CD146 in CRC progression.