The kinetic mechanisms of two key enzymes in the biotransformation of glycerol to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae, glycerol dehydrogenase (GDH) and 1,3-propanediol oxidoreductase (PDOR), was characterized. Kinetics on initial velocity and product inhibition revealed that GDH and PDOR follow an ordered Bi-Bi sequential mechanism. Kinetic models for GDH and PDOR showed that the oxidation reaction catalyzed by GDH was the rate-limiting step in coupled enzymatic reaction when the GDH/PDOR was 1:1, and the NAD+ was the main form of coenzyme in the reaction. Knowledge about the kinetic mechanisms will be helpful to understand how these enzymes is regulated, which will be useful for further enzyme catalysis and metabolic engineering studies.