The differentiation and lipid metabolism of preadipocytes are crucial processes in IMF deposition. Studies have demonstrated that SIRT4 plays essential roles in energy metabolism and redox homeostasis, with its expression being coordinately regulated by multiple transcription factors associated with energy and lipid metabolism. In this study, the findings of multiple omics analysis reveal that SIRT4 significantly up-regulates the expression of genes involved in adipogenesis and enhances the differentiation and lipid deposition of bovine preadipocytes. Furthermore, SIRT4 profoundly influences the expression pattern of metabolites by increasing the abundance of substances involved in lipid synthesis while decreasing those that promote lipid oxidative decomposition. Additionally, SIRT4 broadly up-regulates the expression levels of various lipid classes, including glycerolipids, glycerophospholipids, sphingolipids, and sterol lipids. These findings not only provide a theoretical basis for molecular breeding and genetic improvement in beef cattle, but also offer potential therapeutic approaches for energy homeostasis disorders and obesity.