MicroRNAs (miRNAs) are endogenous 21-24 nucleotide single-stranded RNAs and play important roles in gene expression, development, cell cycles and environmental stresses.Plant miRNAs down-regulate the abundance of their target mRNAs by post-transcription cleavage.Anthocyanin gives plants a colorful world exhibiting from red, blue to purple and play an importantrole in pollination, seed dispersal, and stress resistance.Particularly, being antioxidant activity, anthocyanin is one of key features of nutrition quality in fruits and vegetables.Although some transcription factors involved in the anthocyanin biosynthetic pathwayhave been well-documented, regulatory mechanisms underlying anthocyanin biosynthesisare still not fully understood.In this study, we demonstrated that anthocyanin biosynthesis was finely regulated by multiple miRNAs includingmiR828, miR858, miR165/166 and miR156 in fruit of tomato(Solanum lycopersicum-Ailsa Graig), the most popular vegetable in the world.Bioinformatics analysis predicted the target genes for miR828 areTrans-Acting SiRNA Gene 4 (TAS4), Mby-like1 and EIN2.Mby-like1 involves in regulation of anthocyanin biosynthesis in tomato while EIN2 is responsible for fruit development.MiR165/166 targets genes of the class Ⅲhomeodomain leucine-zipper (HD-ZIP Ⅲ).The transcriptional factor HD-ZIP Ⅲ controls multiple biological aspects such plant tissue pattern and polarity.MiR156 was found to target squamosal promoter binding protein-like 9(SPL9) gene which regulate phase change, flowering and anthocyanin accumulation.miR858 targets MYB 12, MYB 15 and several other myb-like genes.Over-expression vectors (expressing pri-miRNAs) and reduced-expression vectors (a short tandem targetmimic, STTM) were constructed separately and were transferred into tomato (cv.Ailsa Craig) by Agrobacterium tumefaciena-mediated approach respectively.For the pri-miRNA-overexpressed plants, anthocyanin accumulation reduced by 3-5 folds compared to the WT plants.In contrast, anthocyanin content was increased by 4-7 folds in the STTM transgenic plants (the interest miRNA expression was reduced by 95% at least).Expression analysis indicated that the interest matured mRNA expressions were enhanced greatly in the over-expressed plants whereas their expressions were almost blocked in the STTM transgenic plants.Correspondingly, the target genes of miR828, such as Mby-like1 and EIN2, reduced in the pri-miR828 overexpression plants, but increased largely in the STTM828 transgenic plants.Similarly, MiR156-target gene SPL9 expression was decreased in the pri-MiR156-overexpressed plant, and enhanced in the STTMmiR156 transgenic plants.For miR165/166, the expressions of all its five HD-ZIP Ⅲ target genes were changed cordately in either pri-miR165/166 over-expressers or STTMmiR165/166 transgenics.However, the expression changes of the HD-ZIP Ⅲ mRNAs were not as dramatic in the transgenic tomatoes as they expressed in the transgenic Arabidopsis.Therefore, phenoptypic variations in growth and development were not significant in the transgenic tomatoes while the variations were very strong in the transgenic Arabidopsis.It looks like that there may be different factors responsible for miR165/166-meadiated regulation of its target gene HD-ZIP Ⅲ between tomato and Arabidopsis.Following by expression changes of those target genes of the miRNAs, the corresponding changes were detected in the transgenic plants for the transcriptional levels of genes including PAL, PAP1, CHS, CHI, F3H F3 H and DFR that directly guide anthocyanin biosynthesis.Our findings suggest that multiple miRNAs modulate anthocyanin biosynthesis in tomato plants.Weproposed a model to explain the regulatory network mediated by miR828, miR858, miR165/166 and MiR156 in anthocyanin biosynthesis pathway.