Aims: The purpose of the present study was to determine whether miR-21 regulates the human ACAT1 gene. We also assessed whether transfection of MCF-7 cells with miR-21 mimic/inhibitor leads to changes in ACAT1 mRNA/protein levels, cell proliferation rate, or apoptosis. Main methods: Regulation of ACAT1 3′UTR by miR-21 was evaluated using a dual-luciferase reporter assay. The effect of miR-21 on mRNA/protein levels of ACAT1 and PTEN (confirmed as an important target of miR-21 for comparison) was measured by qPCR/western blot analysis and immunostaining. Proliferation rate was determined by cell counting. Percentage of cells undergoing late apoptosis was determined by staining with Hoechst 33342/propidium iodide. Key findings: Dual-luciferase reporter assay confirmed the regulation of ACAT1 3′UTR by miR-21. Furthermore, transfection of MCF-7 cells with miR-21 mimic decreased mRNA and protein levels of ACAT1 and PTEN genes. In contrast, miR-21 inhibition increased the mRNA and protein levels of both genes studied. Finally, we observed an increase in cell proliferation and decrease in the percentage of cells in late apoptosis in MCF-7 cells transfected with miR-21 mimic, whereas transfection with miR-21 inhibitor led to the opposite effect. Significance: Our data confirm the hypothesis that miR-21 regulates the human ACAT1 gene. As the expression of this microRNA is altered in many types of cancers, the discovery of novel targets for miR-21 is of particular interest for diagnosis and treatment.