Repeated batch cultivation (empty-and-fill protocol) using obligate anaerobe Clostridium diolis was attempted in the present study to improve the production of 1,3-propanediol (1,3-PD). In repeated batch operation, 20 % (v/v) culture broth was removed from the bioreactor and supplemented with an equal volume of fresh nutrient medium when the residual glycerol concentration in the bioreactor decreased below 15 g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8 g/L with a productivity of 1.04 g/L/h at the end of 65 h. This represented a 1,3-PD concentration and productivity enhancement by 2.6-fold and 1.5-fold, respectively, as compared to batch 1,3-PD fermentation. This is the first report on the use of repeated batch mode of bioreactor operation for enhanced 1,3-PD production.