Abstract: This study aims to investigate the dynamic process of amino acid metabolism in the Clostridium acetobutylicum fermentation of biobutanol. In a production strategy, a type of cheap nitrogen source, such as amino acid in the Corn Steep Liquor (CSL), can be used to produce butanol from Clostridium acetobutylicum. The efficient and cheap nitrogen source is seeking for butanol production to reduce the production cost of fermentation. In this study, a high-throughput sequencing technology was used to analyze the microbial diversity in CSL, based on the dynamic model of carbon metabolism for ABE (acetone-butanol-ethanol) produce by Clostridium acetobutylicum. A model of amino acid metabolism was constructed, using the metabolism network of amino acid, chemical reaction flux dynamics, and mass balance measurement. 15 amino acids were selected to simulate the metabolism process in ABE fermentation, using xylose as a carbon source. A redundancy analysis was conducted to establish the correlation between amino acid metabolism, the biomass of Clostridium acetobutylicum, and the synthesis of ABE. The predict model was achieved for the consumption process of amino acid in the CSL fermentation in an actual production. The results showed that the Clostridium accounted for 68.76% of the total number of bacteria, indicating the dominant bacterial group in the CSL. The prediction results showed that the gene function of original bacteria was mainly focused on the transformation of carbon and nitrogen sources in the CSL. The optimal parameter correction demonstrated that the simulated values were in good agreement with the experimental ones, indicating that the proposed model can accurately reveal the dynamic metabolic process of amino acid under the utilization by acetobutylicum. Both experimental and simulation results indicated that 11 amino acids (phenylalanine, threonine, isoleucine, leucine, methionine, valine, tyrosine, glycine, serine, arginine, asparagine) were rapidly consumed for cell growth and solvent generation during the cultivation process, whereas 3 amino acids (proline, histamine, aspartic acid) remained stable, particularly that the glutamine accumulation was detected during fermentation. In addition, the metabolism of sugar and amino acid can be beneficial to the cell growth in the early stage, but not to the butanol synthesis. Correlation analysis results showed that five types of amino acids (serine, asparagine, glycine, leucine, and valine) were closely related to the fermentation products and biomass accumulation. The correlation sequence was serine, glycine, leucine, valine, asparagine in order from the largest to the smallest. A high consumption rate was obtained for valine, glycine, and serine during the fermentation process, indicating a nutrient limiting factor in the later stage of fermentation. A fully consumption was found in the predicted values of valine, glycine, and serine in the fermentation of CSL, indicating that the C. acetobutylicum had the highest utilization rate of three amino acids. Therefore, amino acids can be expected to add in the late period of fermentation, to avoid the deficiency of nutrient limiting factors. The CSL can also serve as the dominant nitrogen source of butanol fermentation by Clostridium The findings can provide a sound theoretical reference and data support for the amino acid metabolism of Clostridium acetobutylicum, and for the subsequent utilization and optimization of CSL as a promising nitrogen source to produce biobutanol.