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microRNAs (miRNAs) play critical regulatory roles in plant growth and development. In the present study, the function of herbaceous peony (Paeonia lactifloraPall.) miR156e-3p in the regulation of color formation has been investigated. Firstly, P. lactifloramiR156e-3p precursor sequence (pre-miR156e-3p) was isolated. Subsequently, the overexpression vector of pre-miR156e-3p was constructed and transformed into Arabidopsis thaliana. Moreover, the medium screening, GUS staining, polymerase chain reaction (PCR) of the GUS region and real-time quantitative PCR (qRT-PCR) of miR156e-3p all confirmed that the purpose gene had been successfully transferred into Arabidopsisplants and expressed, which resulted in apparent purple lateral branches. And this change in color was caused by the improved anthocyanin accumulation. In addition, expression analysis had shown that the level of miR156e-3p transcript was increased, while transcription level of target gene squamosa promoter binding protein-like gene (SPL1), encoding SPLtranscription factor that negatively regulated anthocyanin accumulation, was repressed in miR156e-3p-overexpressing transgenic plants, and its downstream gene dihydroflavonol 4-reductase gene (DFR) that was directly involved in anthocyanin biosynthesis was strongly expressed, which resulted in anthocyanin accumulation of Arabidopsislateral branches. These findings would improve the understanding of miRNAs regulation of color formation in P. lactiflora.