The Wnt1 protein, a secreted ligand that activates Wnt signaling pathways, contributes to the self-renewal of cancer stem cells (CSCs) and thus may be a major determinant of tumor progression and chemoresistance. In a series of gastric cancer specimens, we found strong correlations among Wnt1 expression, CD44 expression, and the grade of gastric cancer. Stable overexpression of Wnt1 increased AGS gastric cancer cells' proliferation rate and spheroids formation, which expressed CSC surface markers Oct4 and CD44. Subcutaneous injection of nude mice with Wnt1-overexpressing AGS cells resulted in larger tumors than injection of control AGS cells. Salinomycin, an antitumor agent, significantly reduced the volume of tumor caused by Wnt1-overexpressing AGS cells in vivo. This is achieved by inhibiting the proliferation of CD44+Oct4+ CSC subpopulation, at least partly through the suppression of Wnt1 and β-catenin expression. Taken together, activation of Wnt1 signaling accelerates the proliferation of gastric CSCs, whereas salinomycin acts to inhibit gastric tumor growth by suppressing Wnt signaling in CSCs. These results suggest that Wnt signaling might have a critical role in the self-renewal of gastric CSCs, and salinomycin targeting Wnt signaling may have important clinical applications in gastric cancer therapy.