This study presents an analysis of metabolic fluxes of the anaerobic glycerol metabolism by Klebsiella pneumoniae for the production of 1,3-propanediol (1,3-PD). A metabolic network was reconstructed from the partly annotated genome sequence as well as from biochemical and physiological literatures. Extracellular fluxes measured in continuous culture under steady condition were used to estimate intracellular fluxes. A comparison of changes in the branch point of 3- hydroxypropionaldehyde (3-HPA) flux distribution reveals that glycerol dehydratase (GDHt) is the rate-limiting enzyme at high glycerol concentration and 1,3-propanediol oxidoreductase (PDOR) is the rate-limiting enzyme at low initial glycerol concentration for the production of 1,3-PD due to an insufficient supply of reducing equivalents.