Bacterial nanocellulose (BNC) is a valuable biopolymer with immense potential in various sectors of biotechnology. However, large-scale production is hindered by low yields and high costs. Glycerol is an inexpensive and widely available carbon source for BNC biosynthesis, as it is a by-product of the biofuel industry. Compared to glucose, this polyol enhances BNC yields of Novacetimonas hansenii SI1 and related strains. This study investigates transcriptomic changes in N. hansenii SI1 after switching from glucose to glycerol using RNA-seq. The results reveal metabolic reprogramming, including upregulation of genes involved in glycerol uptake and catabolism, gluconeogenesis, the pentose phosphate pathway, and the Entner-Doudoroff pathway. Glycerol metabolism induces oxidative stress, evidenced by elevated expression of antioxidant enzymes, repair proteins, and metal ion homeostasis systems. Additionally, pathways such as riboflavin biosynthesis, methionine salvage, and sulphur assimilation are upregulated to mitigate oxidative damage. Increased oxidative conditions likely stimulate c-di-GMP synthesis, activating cellulose synthase and promoting BNC production. Furthermore, the acetan-like polymer biosynthetic pathway is significantly induced, further enhancing BNC yield. These findings expand our understanding of glycerol utilisation in BNC production, supporting cost-efficient and eco-friendly processes for maximising biopolymer exploitation. KEY POINTS: • Growth on glycerol remodels central carbohydrate metabolism • Glycerol metabolism induces oxidative stress • Acetan-like biosynthesis and posttranslational effects stimulate BNC production.