Cancer stem cells (CSCs) possess the ability of self-renewal and tumor initiation. Targeting key signaling pathways that are active in CSC self-renewal is one approach to cancer therapy. Abnormal activation of the Wnt/β-catenin pathway has been described in a wide variety of human cancers and in CSCs; however, the role of this pathway in gastric CSCs has not been reported. In our study, we investigated whether the Wnt/β-catenin pathway plays an important role in gastric CSCs. First, we isolated cancer stem-like cells (CSLCs) from the human gastric cancer cell line MKN-45 using tumorsphere cultures. We tested whether tumorsphere cells were CSLCs using the following three criteria: i) We identified that the expression of the CSC marker CD44 was significantly greater in tumorsphere cells compared to adherent cells; ii) compared with adherent cells, the floating tumorsphere cells had greater self-renewing capacity; iii) in vivo xenograft studies showed that tumorsphere cells generate larger tumors than adherent cells at the same number. In addition, we studied the mechanism(s) by which the canonical Wnt signaling pathway acts in CSLCs. Western blotting and real-time PCR showed that the expression levels of β-catenin and c-myc, cyclin d1 and axin 2 were downregulated/upregulated with the inhibition/activation of the Wnt pathway. The pathway blocked by DKK-1 caused a higher reduction in the self-renewing capacity of MKN-45 tumorsphere cells and the pathway activated by lithium chloride improved the self-renewal of CSLCs. In conclusion, our data suggested that the Wnt/β-catenin pathway is essential for the self-renewal of CSLCs in human gastric cancer.