Abstract: Soybean oil is an important carbon source for the efficient synthesis of secondary metabolites. In this paper, a novel feeding strategy of glucose and soybean oil combination was developed, which significantly improved the synthesis efficiency of Cephalosporin C (CPC) by 14.8%. A multi-scale research on the mechanism for CPC enhancement revealed that the combination of carbon source combination globally changed cell respiration metabolism, intermediate metabolites pool size, metabolic flux distribution, and NADPH utilization. The significant change in carbon dioxide evolution rate (CER) indicated a significant alteration in central metabolic flux under the glucose and soybean oil combination feeding strategy, leading to strengthened carbon flux for CPC synthesis and increased extracellular absorption rates of methionine and serine. Intracellular pools of α- ketoglutarate, pyruvate, and malate were significantly increased, which were beneficial for core amino acids synthesis such as glutamic acid, valine, and cysteine to facilitate CPC production. The loading analysis of principal component analysis (PCA) showed that the most of amino acids positively influenced CPC synthesis, and intracellular glutamic acid content was found to have the strongest positive correlation. However, the accumulation of α-aminoadipic acid and lysine was unfavorable for CPC synthesis. In terms of reducing power, the combination of soybean oil and glucose significantly alleviated NADPH competition between soybean oil utilization and CPC synthesis, thus meeting high demand for rapid CPC synthesis. These results indicated that substrate changes presented a comprehensive impact on cellular metabolism of cells, reflecting the great potential of this strategy in optimizing the CPC production efficiency.