Genistein is an isoflavone from soy with multiple action targets in cellular processes. Hedgehog signaling and its activator Gli1 are involved not only in oncogenesis, but also in cancer stemness and overexpression of CD44, a typical cancer stem cell surface marker. It has been shown that levels of Gli1 and CD44 expression are downregulated by genistein. Genistein may modulate distinctive cellular characteristics in cancer stem cells by inhibiting Gli1-related signaling pathways. In the present study, we sorted cells from MKN45, a human gastric cancer cell line, according to CD44 expression. CD44(+) cells showed properties of cancer stem-like cells and formed sphere colonies. In addition, sonic hedgehog (Shh) signaling genes were upregulated in CD44(+) cells when compared with these levels in CD44(-) cells. When CD44(+) cancer stem-like cells were treated with genistein, Gli1 and CD44 mRNA and protein expression was significantly reduced. Moreover, other stem cell markers were downregulated by genistein. Gli1 siRNA was used to confirm the action of genistein in inhibiting Gli1 expression. The high cell migration capacity of CD44(+) cells was blocked by genistein. in conclusion, genistein inhibits Gli1 gene expression, resulting in the attenuation of cancer stem-like properties in gastric cancer cells. In addition, genistein suppresses the cell invasive capacity that is required for tumor growth and metastasis. Our data showed that genistein can be an effective agent for gastric cancer therapy by targeting cancer stem-like characteristics.