The prognosis of advanced or recurrent colorectal cancer is still poor. Dye-effluxing side population (SP) colon cancer cells are reportedly resistant to chemotherapeutic agents. Most sporadic colorectal cancers involve constitutive activation of the Wnt signaling pathway. In this study, we examined the effect of the Wnt signaling on SP cells and the possibility that inhibition of Wnt signaling may decrease the resitance to chemotherapeutic drugs in the human colon cancer cells. Drug resistance of SP cells to 5-fluorouracil (5-FU) and irinotecan, decrease of SP cells by the Wnt signaling inhibition and activation of the Wnt signaling of the sorted SP cells were examined using the SW480 colon cancer cell line. mRNA expressions of ATP-binding cassette (ABC) transporters when Wnt signaling was inhibited were evaluated with real-time PCR using colon cancer cell lines (SW480, DLD-1, HCT116, HT29 and LOVO). The sensitivity to irinotecan and paclitaxel when the Wnt signaling was inhibited was investigated using SW480. Inhibition of Wnt signaling was performed by siRNA of β-catenin. SP cells showed more resistance to 5-FU and irinotecan, and higher activation of the Wnt signaling pathway, than non-SP cells. Silencing of β-catenin decreased significantly more SP cells than non-SP cells. Expression of ABC transporter genes, such as ABCB1 and ABCG2, was significantly higher in SP cells than non-SP cells. Silencing of β-catenin decreased transcription of these ABC transporter genes; β-catenin-silenced cells became relatively sensitive to paclitaxel and irinotecan. These results indicate that inhibiting the Wnt signaling pathway may be a fruitful strategy for targeting chemotherapy-resistant colon cancer cells, including SP cells.