In recent years, glycerol has become an attractive carbon source for microbial processes, as it accumulates massively as a by-product of biodiesel production, also resulting in a decline of its price. A potential use of glycerol in biotechnology is the synthesis of poly(3-hydroxypropionate) [poly(3HP)], a biopolymer with promising properties which is not synthesized by any known wild-type organism. In this study, the genes for 1,3-propanediol dehydrogenase (dhaT) and aldehyde dehydrogenase (aldD) of Pseudomonas putida KT2442, propionate-coenzyme A (propionate-CoA) transferase (pct) of Clostridium propionicum X2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha H16 were cloned and expressed in the 1,3-propanediol producer Shimwellia blattae. In a two-step cultivation process, recombinant S. blattae cells accumulated up to 9.8% ± 0.4% (wt/wt [cell dry weight]) poly(3HP) with glycerol as the sole carbon source. Furthermore, the engineered strain tolerated the application of crude glycerol derived from biodiesel production, yielding a cell density of 4.05 g cell dry weight/liter in a 2-liter fed-batch fermentation process.