Towards a global objective to produce chemical derivatives by microbial processes, this work dealt with a metabolic engineering of the yeast Saccharomyces cerevisiae for glycerol production. To accomplish this goal, overexpression of GPD1 was introduced in a tpi1delta mutant defective in triose phosphate isomerase. This strategy alleviated the inositol-less phenotype of this mutant, by reducing the levels of dihydroxyacetone phosphate and glycerol-3-P, two potent inhibitors of myo-inositol synthase that catalyzes the formation of inositol-6-phosphate from glucose-6-phosphate. Further deletion of ADH1 and overexpression of ALD3, encoding, respectively, the major NAD+-dependent alcohol dehydrogenase and a cytosolic NAD+-dependent aldehyde dehydrogenase yielded a yeast strain able to produce 0.46 g glycerol (g glucose)(-1) at a maximal rate of 3.1 mmol (g dry mass)(-1) h(-1) in aerated batch cultures. At the metabolic level, this genetic strategy shifted the flux control coefficient of the pathway to the level of the glycerol efflux, with a consequent intracellular accumulation of glycerol that could be partially reduced by the overproduction of glycerol exporter encoded by FPS1. At the transcriptomic level, this metabolic reprogramming brought about the upregulation of genes encoding NAD+/NADP+ binding proteins, a partial derepression of genes coding for TCA cycle and respiratory enzymes, and a downregulation of genes implicated in protein biosynthesis and ribosome biogenesis. Altogether, these metabolic and molecular alterations stand for major hurdles that may represent potential targets for further optimizing glycerol production in yeast.