Cancer initiating cells (CIC) are defined as the unique subpopulation in the tumors that possess the ability to initiate tumor growth and sustain self-renewal as well as metastatic potential. In this study, we found that EHF overexpression promoted formation of CIC traits and silencing it inhibited the traits in gastric cancer NCI‑N87 cells. Overexpressing EHF downregulated the antitumor effect of 5-fluorouracil (5-FU) in NCI‑N87 cells. We found that miR‑206 downregulated EHF protein expression by targeting its 3'UTR in NCI‑N87 cells and GES-1 cells. Overexpressing miR‑206 inhibited formation of CIC in NCI‑N87 cells. In gastric cancer tissues, EHF protein expression was upregulated and miR‑206 was downregulated. We identified a negative correlation between EHF protein and miR‑206 expression in gastric cancer tissues. Thus, we concluded that miR‑206 inhibits formation of CICs by targeting EHF in gastric cancer.