The microbial conversion of glycerol into value-added commodity products has emerged as an attractive means to meet the demands of biosustainability. However, glycerol is a non-preferential carbon source for productive fermentation because of its low energy density. We employed evolutionary and metabolic engineering in tandem to construct an Escherichia coli strain with improved GABA production using glycerol as the feedstock carbon. Adaptive evolution of E. coli W under glycerol-limited conditions for 1300 generations harnessed an adapted strain with a metabolic system optimized for glycerol utilization. Mutation profiling, enzyme kinetic assays, and transcriptome analysis of the adapted strain allowed us to decipher the basis of glycerol adaptation at the molecular level. Importantly, increased substrate influx mediated by the mutant glpK and modulation of intracellular cAMP levels were the key drivers of improved fitness in the glycerol-limited condition. Leveraging the enhanced capability of glycerol utilization in the strain, we constructed a GABA-producing E. coli W-derivative with superior GABA production compared to the wild-type. Furthermore, rationally designed inactivation of the non-essential metabolic genes, including ackA, mgsA, and gabT, in the glycerol-adapted strain improved the final GABA titer and specific productivity by 3.9- and 4.3-fold, respectively, compared with the wild-type.