Glycerol is considered as an ideal feedstock for producing bioplastics via bacterial fermentation due to its ubiquity, low price, and high degree of reduction substrate. In this work, we study the yield and cause of limitation in poly(3-hydroxybutyrate) (PHB) production from glycerol. Compared to glucose-based PHB production, PHB produced by Cupriavidus necator grown on glycerol has a low productivity (0.92 g PHB/L/h) with a comparably low maximum specific growth rate of 0.11 h−1. We found that C. necator can synthesize glucose from glycerol and that the lithotrophical utilization of glycerol (non-fermentative substrate) or gluconeogenesis is an essential metabolic pathway for biosynthesis of cellular components. Here, we show that gluconeogenesis affects the reduction of cell mass, the productivity of biopolymer product, and the molecular chain size of intracellular PHB synthesized from glycerol by C. necator. We use NMR spectroscopy to show that the isolated PHB is capped by glycerol. We then characterized the physical properties of the isolated glycerol-based PHB with differential scanning calorimetry and tensile tests. We found that although the final molecular weight of the glycerol-based PHB is lower than those of glucose-based and commercial PHB, the thermal and mechanical properties of the biopolymers are similar. Biotechnol. Bioeng. 2012; 109: 2808–2818. © 2012 Wiley Periodicals, Inc.