The production of 1,3-propanediol (1,3-PDO) from glycerol was studied by GRAS and native 1,3-PDO producer, Lactobacillus reuteri DSM 20016. This strain ferments glucose with production of lactate, acetate, ethanol, and converts glycerol to 1,3-PDO using NADH generated by glucose metabolism. To improve 1,3-PDO production, alcohol dehydrogenases (ADH) were disrupted and 1,3-PDO oxidoreductases (PDOR) were overexpressed. Deletion of ADH (adh2) enhanced 1,3-PDO production yield on glucose by reducing ethanol synthesis, and overexpression of PDOR (pduQ) elevated 1,3-PDO production rate and cell growth rate. The strain with simultaneous adh2 deletion, pduQ overexpression (Delta adh2pduQ++) could produce 687 mM 1,3-PDO with the yield of 1.2 +/- 0.08 mol 1,3-PDO/mol glucose by fed-batch bioreactor cultivation in 48 h. However, the 1,3-PDO production rate was greatly reduced in the late period of bioreactor culture, mainly due to high lactate accumulation. This is the first report on rational metabolic engineering of L. reuteri for improved 1,3-PDO production.