Background:MiRNAs are significant regulatory factors in stem cell proliferation, and alteration in miRNA expression influences the viability and gene expression of cancer stem cells. Herein, we evaluated the effect of the hsa-miR-4270 inhibitor and mimic on the expression of stem cell markers in GCSCs.Methods:GCSCs were isolated from the MKN-45 cell line by a non-adherent surface system. The cells were confirmed by differentiation assays using dexamethasone and insulin as adipogenesis-inducing agents and also staurosporine as a neural-inducing agent. Isolated GCSCs were treated with different concentrations (0, 15, 20, 25, 30, 40, 50, and 60 nM) of hsa-miR-4270 inhibitor and mimic. The cell viability was determined by Trypan blue method. Transcription of the stem cell marker genes, includingCD44,OCT3/4,SOX2,Nanog, andKLF4, was evaluated by quantitative real-time RT-PCR.Results:The results showed that GCSCs were differentiated into adipose cells using dexamethasone and insulin, and neural cells by staurosporine. Treatment of the GCSCs with hsa-miR-4270 inhibitor decreased the cell viability and downregulatedOCT3/4,CD44, andNanoggenes to 86%, 79%, and 91% respectively. Also,SOX2andKLF4were overexpressed to 8.1- and 1.94-folds, respectively. However, hsa-miR-4270 mimic had opposite effects on the cell viability and gene expression of the stem cell markers.Conclusion:Effect of hsa-miR-4270 inhibitor and mimic on the expression of the stem cell markers in GCSCs indicates that hsa-miR-4270 stimulates the stemness property of GCSCs, likely through stimulating the development of gastric stem cells.