Backgroud: The Distal-less 3 (DLX3) is a Distal-less homeodomain protein that belongs to the members of the DLX vertebrate family.DLX3 acts as a transcriptional activator and plays a critical role in the development of hair follicle and hair formation.Previously, the four SNPs in 3UTR of sheep DLX3 gene and their haplotypes had consistently significant effect on wool curvature trait.Additionally, luciferase reporter assay indicated that these four SNPs all have a functional role for the expression of DLX3 gene.Methods: The objective of this study was to deeply explore the effect mechanisms of the four SNPs in 3UTR of sheep DLX3 gene.The online bioinformatics software was applied to analyze the sequence structure and motifs in 3UTR, and the binding change of miRNAs caused by single nucleotide mutation in 3UTR of sheep DLX3 gene.Then, we used luciferase reporter assay and Real-time PCR to validate the results of bioinformatics prediction.Results: The bioinformatics analysis showed that the SNP function in 3UTR of the sheep DLX3 gene was mediated by the miRNA.We finally confirmed that the miR-204*, miR-49 l-5p and the miR-188 targeted into the 3UTR of sheep DLX3 gene, and the regulation of these miRNAs were affected by the SNP c.*ll8T>C, c.*228T>C, c.*1038_1039 ins1, respectively.And the mRNA expression level of DLX3 gene and its target genes downstream were changed for these miRNAs regulation.Conclusions: All the four SNPs in 3UTR of sheep DLX3 gene were functional sites, and they played roles via regulation of miRNA at least.The current study laid the foundation for the probe into the correlation between the polymorphisms in 3UTR of sheep DLX3 gene and wool curvature trait .