1,3-Propanediol (1,3-PD), an important material for chemical industry, is biologically synthesized by glycerol dehydratase (GDHt) and 1,3-propanediol dehydrogenase (PDOR). In present study, the dhaBCE and dhaT genes encoding glycerol dehydratase and 1,3-propanediol dehydrogenase respectively were cloned from Citrobacter freundii and co-expressed in E. coli. Sequence analysis revealed that the cloned genes were 85 and 77 % identical to corresponding gene of C. freundii DSM 30040 (GenBank No. U09771), respectively. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration, and recombinant GDHt and PDOR were characterized by activity assay, kinetic analysis, pH, and temperature optimization. This research may form a basis for the future work on biological synthesis of 1,3-PD.