AIM To determine the potential roles of CD4 and microRNA (miR)-145 in gastric cancer. METHODS The levels of CD44 and miR-145 were determined in gastric cancer cells. Quantitative real-time polymerase chain reaction was used to measure to the level of CD44 mRNA. A luciferase reporter assay and western blotting were performed to examine the effect of miR-145 on CD44 expression. Tumor sphere and MTT assays were carried out to evaluate the self-renewal and chemo-resistance properties of gastric cancer cells. RESULTS The expression of CD44 was greatly increased and miR-145 was decreased in gastric cancer cells that were highly enriched in cancer stem cells (CSCs). The results demonstrated that miR-145 regulated CD44 by targeting directly the CD44 3'-untranslated region (3'-UTR). In gastric cancer cells, overexpression of miR-145 repressed the activity of the CD44 3'-UTR, and disruption of miR-145/CD44 3'-UTR interactions abrogated the silencing effects. In addition, miR-145 inhibition stimulated CD44 3'-UTR activity and disruption of miR-145/CD44 3'-UTR interactions abrogated this stimulatory effect. Enforced CD44 expression greatly increased tumor sphere formation and chemoresistance in gastric cancer cells. Furthermore, the inhibition of CSCs and the chemo-sensitivity of gastric cancer cells treated with miR-145 were significantly abrogated by overexpression of CD44. CONCLUSION miR-145 targeting of CD44 plays critical roles in the regulation of tumor growth and chemo-resistance in gastric cancer.