Abstract: Biosynthesis of 1,3-propanediol from glycerol by Klebsiella pneumoniae was associated with the consumption of NADH, which was generated/regenerated in the oxidative branch pathway of glycerol in vivo. The availability of NADH would be critical for the final yield of 1,3-propanediol. Formate dehydrogenase gene(fdh) was cloned from Candida boidinii genome and was then transformed into Klebsiella pneumoniae YMU2 by constructed expression vector pMAL TM-p2X-fdh to generate recombinant Klebsiella pneumoniae F-1. Compared with K. pneumoniae YMU2, the yield of 1,3-propanediol and production rate of recombinant F-1 were 78. 6 g·L-1 and 1. 33 g·L-1·h-1, increased by 12. 5% and 41. 2%, respectively. The metabolic flux of strain YMU2 and recombinant F-1 was analyzed with the formation of main metabolites.