Adipose is mainly composed of glycerol and fatty acids. Acyl-CoA medium-chain synthetase 3 is a key enzyme in the first step of fatty acid metabolism. In order to further study the biological function of ACSM3 gene, the complete coding region of ACSM3 gene was connected into pcDNA3.1(+) vector by molecular biological techniques such as cloning, sequencing, double enzyme digestion and ligation. The knockdown plasmid of this gene was constructed by biosynthesis technology. Then, the effect of overexpression / knockdown plasmid of ACSM3 gene was detected in porcine preadipocytes cultured in vitro. The results showed that the overexpression plasmid of ACSM3 gene was successfully constructed, and the overexpression effect of the plasmid could last eight days after transfection. One sequence with significant knockdown effect was selected from three specific interference sequences, and the knockdown effect lasted two days after transfection. The overexpression / knockdown plasmid constructed in this study can provide strong technical support for the further functional research of ACSM3 gene.