In this study, a newly strain named Clostridium butyricum YJH-09 were isolated from the sample of pond soil and identified through physiological, biochemical and 16S rDNA analysis. Then, the dhaT gene encoding a novel 1,3-propanediol dehydrogenase (PDOR) was cloned from this strain and expressed in Escherichia coli BL21(DE3). Subsequently, the recombinant PDOR was purified and the optimal pH and temperature, specific activities and kinetic parameter were investigated. Furthermore, the whole cells of Clostridium butyricum YJH-09 mixed with BL21-dhaT were used to produce 1,3-PD through co-biotransformation. As results, 25.88 g/L of 1,3-PD was generated with 0.54 g/g yield from 50 g/L glycerol in 30 h, and the 1,3-PD production was increased more than 2-fold compared with wild type strain alone. This research would offer useful information for further development of the biosynthesis of 1,3-PD.