Abstract <p><i>Klebsiella pneumoniae</i> is recognized as an efficient producer of 1,3-propanediol. The increase in ventilation rate promoted bacterial growth but inhibited the production of 1,3-propanediol. Bacterial cells were harvested after aeration fermentation at rates of 0 L/min (control group) and 6 L/min (experimental group) for durations of 12, 24, and 48 h. Intracellular metabolites underwent untargeted metabolomics analysis. The results indicate that the down-regulation of 2-oxocarboxylic acid metabolism during the initial phases of fermentation suggests enhanced energy production under high-ventilation fermentation conditions. In contrast, nicotinate and nicotinamide metabolism suggests low oxygen concentration hampers the conversion between NAD⁺ and NADH. The up-regulation of purine metabolism pathways during the mid-term of fermentation suggests that a consistently high-oxygen environment is not conducive to purine metabolism. During the late stage of fermentation, the up-regulation of 3,4-dihydroxyphenylalanine and 5,6-dihydroxyindole-2-carboxylate in the high-ventilation group of Tyr metabolism indicates an enhanced antioxidant capacity of the cell. This study presents metabonomic analysis elucidating the potential mechanism of glycerol fermentation by <i>K. pneumoniae</i> under varied aeration conditions.</p>