Abstract: Obejective To construct miRNA-expressing plasmid vector for interfering the expression of β-catenin in adipose-derived stem cells from Sprague-Dawley rats. Methods The double strands oligonucleotides of miR-expressing cassette were ligated into plasmid pSWH to generate pSWH-miR. Then the PCR product of enhanced green fluorescence protein (EGFP） open reading frame (ORF) fragment was inserted into BamHⅠand XboⅠdigested pSWH-miR to generate pSWH-EGFP-miR. The adipose-derived stemcells from rats (rADSCs) were transfected with pSWH-EGFP-miR respectively. The expression of β-catenin was determined by Western blot at 72 h post-transfection. Results Five miRNA-expressing plasmid vectors were constructed (miR-780, miR-796, miR-1467, miR-1948, miR-1960). miR-780 and miR-796 had the best silencing effect on the expression of β-catenin (P<0.05), less did the miR-1960 (P<0.05), but not the miR-1467 and the miR-1948 (P<0.05). Conclusion miR-780 and miR-796 could interference the β-catenin in ADSCs with high efficiency.