Cancer stem cells are undifferentiated cancer cells that have self‐renewal ability, a high tumorigenic activity, and a multilineage differentiation potential. MicroRNAs play a critical role in regulating gene expression during carcinogenesis. Here, we investigated the role of miR‐7 and the mechanism by which it is dysregulated in gastric cancer stem cells (GCSCs). The stem cell marker, CD44, was used to sort GCSCs by fluorescence‐activated cell sorting. We found that CD44 (+) cells have higher invasiveness and form more number of sphere colonies than CD44 (−) cells. Quantitative real‐time polymerase chain reaction (PCR) revealed that the miR‐7‐5p expression was remarkably downregulated in GCSCs but was significantly increased in the methionine‐deprived medium. The downregulation of miR‐7‐5p results from the increased DNA methylation in the promoter region using the methylation‐specific PCR. Overexpression of miR‐7‐5p reduced the formation of colony and decreased the invasion of GCSCs through targeting Smo and Hes1 and subsequent repressing Notch and Hedgehog signaling pathways in vitro. Notably, upregulating miR‐7‐5p inhibited the growth of tumor in the xenograft model. Hence, these data demonstrated that miR‐7‐5p represses GCSC invasion through inhibition of Smo and Hes1, which provides a potential therapeutic target of gastric cancer treatment.