Background and Objective: CD133-positive colon cancer stem like cells (CSLCs) are resistant to the conventional cytotoxic drug 5-fluorouracil (5-FU). Wnt signaling pathway plays important roles in colon cancer carcinogenesis and metastasis, and regulates the self-renewal capacity of CSLCs. In the present study, we explored the impact of 5-FU on Wnt signaling pathway of CD133-positive colon CSLCs, and the relation between Wnt signaling pathway and drug resistance of CD133-positive colon CSLCs. Methods: Magnetic activation cell separation was used to collect CD133-positive cells from colon cancer cell line DLD1, which was transfected with luciferase reporter for Wnt signaling activity. The activity of Wnt signaling pathway was compared between CD133-positive and CD133-negative cells. After the treatment with 1 μg/mL of 5-FU, the cell proliferation rates of DLD1 cells, CD133-positive cells, and CD133-negative cells were compared. After the treatment with 1 μg/mL and 10 μg/mL of 5-FU for 48 h, Wnt activity was compared between CD133-positive and CD133-negative cells. The expression of CD133 and cell apoptosis of CD133-positive cells was detected after exposure to 50 ng/mL of dickkopf (DKK)-1, a Wnt pathway inhibitor. Results: After the treatment with 5-FU, the cell proliferation rate of CD133-positive cells was higher than that of CD133-negative cells and the sensitivity of CD133- positive cells to 5-FU decreased. Wnt activity was higher in CD133-positive cells than in CD133-negative cells [(46.3± 0.3)% vs. (33.9 ± 2.7)%, P = 0.009]. After the treatment with 1 μg/mL and 10 μg/mL of 5-FU, Wnt activity of CD133- positive cells was (90.1 ± 10.0)% (P = 0.012) and (52.9 ± 2.5)% (P = 0.047), respectively, whereas that of CD133-negative cells was (35.5 ± 3.3)% (P = 0.434) and (26.5 ± 0.4)% (P = 0.046), respectively. CD133 expression in CD133-positive cells decreased from (87.2 ± 5.3)% to (60.6 ± 3.1)% (P = 0.022) after treatment with DKK-1, whereas the cell apoptosis rate increased from (11.8 ± 0.2)% to (28.3 ± 0.6)% (P = 0.013). Conclusions: Wnt activity is higher in CD133-positive DLD1 cells than in CD133-negative DLD1 cells. 5-FU can upregulate Wnt activity of CD133-positive colon CSLCs. Blocking Wnt activity may reverse drug sensitivity of CD133-positive cells to 5-FU.