The glycerol fed-batch fermentations with a novel isolated 1,3-propanediol production strains, designed Klebsiella pneumoniae HR526 were performed in 5 L B. Braun fermenter. The result indicated that 91.47 g/L 1,3-propanediol was produced in 30 h. The extracellular metabolic flux was calculated, indicating PDO flux reached the maximum value in the mid logarithmic growth phase, whereas lactic acid flux reached the maximum value in the stable phase. This article also analyzed the key enzyme activity of PDO synthesis, the maximum PDOR activity was reached in the mid of the logarithmic growth phase, the ratio of the GDHt/PDO and GDHt/GDH is rather higher in the logarithmic growth phase than in the stable, leading to 3-HPA accumulation during the exponential growth phase. The production of PDO is closely related to cell growth.