The production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is limited by synthesis of numerous byproducts. Among them, the accumulation of acetate has the largest negative impact on the fermentation performance. To address the acetate overflow caused by knocking out lactate dehydrogenase, alcohol dehydrogenase and succinate dehydrogenase, several metabolic engineering manipulations were conducted. First, acetate was reduced through enhancing the acetate assimilation pathway by overexpressing heterologous acetyl-CoA synthetase. Then, the polyhydroxybutyrate (PHB) synthesis pathway was introduced to further reprogram the intracellular carbon metabolism. As a result, the best performed strain Kpr-6 produced up to 91.2 g/L extracellular 1,3-PD and 2.56 g/L intracellular PHB which can be easily separated from each other, while the acetate was dramatically reduced. The metabolic engineering strategies developed in this study would be helpful for constructing the microbial cell factory for other similar bio-based chemical production.