Objective: To explore the molecular mechanism of miR-194-5p targeting down regulation of CD44 and inhibiting epithelial mesenchymal transition (EMT) in gastric cancer stem cells (CSCs). Methods: qRT-PCR was used to detect the expression levels of miR-194-5p, CD44, Snail, and membrane matrix metalloproteinase-1 (MT1-MMP) in gastric adenocarcinoma cell lines (SGC-7901, MGC-803, SPAG-9, and MNK-45) and gastric mucosal cells (GES-1). Western blot was used to detect the expression levels of CD44, Snail, and MT1-MMP proteins. miR-194-5p over-expression and low-expression plasmid vectors were constructed in vitro then to transfect with lentivirus. The experiment was divided into over-expression group, control group, and low-expression group. qRT-PCR and Western blot were used to detect changes of miR-194-5p, CD44, Snail, and MT1-MMP; CCK8 assay was used to detect cell proliferation rate, flow cytometry was used to detect cell apoptosis rate, Transwell assay was used to detect cell invasiveness, and Western blot was used to detect the expression levels of EMT markers E-cadherin and N-cadherin proteins. Results: The expression of miR-194-5p was significantly lower in gastric adenocarcinoma cell lines than gastric mucosal cells, while the expression levels of CD44, Snail, and MT1-MMP were significantly higher; what's more, the expression differences of SGC-7901 and MGC-803 compared to gastric mucosal cells were the most (P<0.05). Compared with the control group, miR-194-5p expression in the over-expression group was significantly more, but CD44, Snail, and MT1-MMP expressions were significantly less, cell proliferation and invasiveness were lower, apoptosis rate was higher, E-cadherin was upregulated, and N-cadherin was downregulated (P<0.05). Compared with the control group, the expression level of miR-194-5p was significantly lower in the low-expression group, while the expression levels of CD44, Snail, and MT1-MMP were significantly higher, the cell proliferation rate and invasiveness were more, while the apoptosis rate was less, E-cadherin was down-regulated, and N-cadherin was upregulated (P<0.05). Conclusion: Low expression of miR-194-5p in gastric cancer may exert an anti-tu-mor effect by downregulating the expression of CD44 in CSCs, thereby inhibiting the occurrence of EMT. [ABSTRACT FROM AUTHOR]