Simple SummaryTriacylglycerol (TAG) is the primary component of intramuscular fat, an important factor in determining meat quality. The synthesis of TAG is regulated by diacylglycerol o-acyltransferase (DGAT). This study investigated the regulatory mechanisms of two subtypes ofDGAT, namelyDGAT1andDGAT2, in the differentiation of Yanbian bovine preadipocytes and their roles in lipid metabolism-related signaling pathways. sh-DGAT1 and sh-DGAT2 were prepared using short interfering RNA (siRNA) interference technique targetingDGAT1andDGAT2genes and infected with bovine preadipocytes separately or simultaneously. Differentially expressed genes (DEGs) in bovine preadipocytes were analyzed using RNA sequencing and genome databases. During bovine preadipocytes differentiation, interference withDGAT1andDGAT2inhibited the formation of lipid droplets, content, and expression of lipid-forming genes at the mRNA level.DGAT2showed a stronger inhibitory effect. Transcriptome analysis revealed 2070 and 2242 DEGs between pre-adipocytes and normal cells that inhibitedDGAT1andDGAT2expression, respectively, and 2446 DEGs when both were simultaneously inhibited. Our results indicate that compared withDGAT1,DGAT2plays a more important role in regulating bovine fat metabolism, which provides a theoretical basis for producing high-quality marbled beef.AbstractTriacylglycerol (TGA) is the primary component of intramuscular fat. Expression of diacylglyceryl transferase (DGAT) determines the polyester differentiation ability of precursor adipocytes. The twoDGATisoforms (DGAT1andDGAT2) play different roles in TAG metabolism. This study investigates the roles ofDGAT1andDGAT2in signaling pathways related to differentiation and lipid metabolism in Yanbian bovine preadipocytes. sh-DGAT1 (sh-1), sh-DGAT2 (sh-2), and sh-DGAT1 + sh-DGAT2 (sh-1 + 2) were prepared using short interfering RNA (siRNA) interference technique targetingDGAT1andDGAT2genes and infected bovine preadipocytes. Molecular and transcriptomic techniques, including differentially expressed genes (DEGs) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis, were used to investigate the effects on the differentiation of Yanbian bovine preadipocytes. After interference withDGAT1andDGAT2genes, the contents of TAG and adiponectin were decreased. The TAG content in the sh-2 and sh-1 + 2 groups was significantly lower than that in the sh-NC group. RNA sequencing (RNA-seq) results showed 2070, 2242, and 2446 DEGs in the sh-1, sh-2, and sh-1 + 2 groups, respectively. The DEGs of the sh-2 group were mainly concentrated in thePPAR,AMPK, andWntsignaling pathways associated with adipocyte proliferation and differentiation. These results demonstrated that at the mRNA level,DGAT2plays a more important role in lipid metabolism thanDGAT1.