The demand for 1,3-propanediol (1,3-PDO) has increased sharply due to its role as a monomer for the synthesis of polytrimethylene terephthalate (PTT). AlthoughClostridium butyricumis considered to be one of the most promising bioproducers for 1,3-PDO, its low productivity hinders its application on industrial scale because of the longer time needed for anaerobic cultivation. In this study, an excellentC. butyricum(DL07) strain was obtained with high-level titer and productivity of 1,3-PDO, i.e., 104.8 g/L and 3.38 g/(L center dot h) vs. 94.2 g/L and 3.04 g/(L center dot h) using pure or crude glycerol as substrate in fed-batch fermentation, respectively. Furthermore, a novel sequential fed-batch fermentation was investigated, in which the next bioreactor was inoculated byC. butyricumDL07 cells growing at exponential phase in the prior bioreactor. It could run steadily for at least eight cycles. The average concentration of 1,3-PDO in eight cycles was 85 g/L with the average productivity of 3.1 g/(L center dot h). The sequential fed-batch fermentation could achieve semi-continuous production of 1,3-PDO with higher productivity than repeated fed-batch fermentation and would greatly contribute to the industrial production of 1,3-PDO byC. butyricum.