1,3-propanediol is an important chemical widely used in polymer production. In this study, two strains, Zygosacharomyces rouxii JL2011 and Klebsiella pneumoniae S6, were used as a mixed culture for 1,3-propanediol production directly from glucose. Two important parameters including inoculation time of K. pneumoniae S6 at stage of mixed culture and initial cell ratio of Z. rouxii JL2011 to K. pneumoniae S6 in mixed fermentation were optimized in culture flasks. In those experiments, the best results were obtained with a yield of 6.8 g/L 1,3-propanediol from glucose when K. pneumoniae S6 was inoculated after 48 h in the culture of Z. rouxii JL2011 by mixed culture of Z. rouxii JL2011 and K. pneumoniae S6 with initial cell ratio of 1:200. In a 7-L bioreactor, the maximum 1,3-propanediol production could reach up to 15.2 g/L. Thus, this study presents an effective process for 1,3-propanediol microbial production from glucose by using mixed culture of Z. rouxii JL2011 and K. pneumoniae S6. This work does not only demonstrate a new way to produce 1,3-propanediol from a low-cost feedstock, but may also make a valuable contribution to the development of a cost-effective fermentation based on renewable resources.