Glycerol is used by the cosmetic, paint, automotive, food, and pharmaceutical industries and for production of explosives. Currently, glycerol is available in commercial quantities as a by-product from biodiesel production, but the purity and the cost of its purification are prohibitive. The industrial production of glycerol by glucose aerobic fermentation using osmotolerant strains of the yeasts Candida sp. and Saccharomyces cerevisiae has been described. A major drawback of the aerobic process is the high cost of production. For this reason, the development of yeast strains that effectively convert glucose to glycerol anaerobically is of great importance. Due to its ability to grow under anaerobic conditions, the yeast S. cerevisiae is an ideal system for the development of this new biotechnological platform. To increase glycerol production and accumulation from glucose, we lowered the expression of TPI1 gene coding for triose phosphate isomerase; overexpressed the fused gene consisting the GPD1 and GPP2 parts coding for glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase, respectively; overexpressed the engineered FPS1 gene that codes for aquaglyceroporin; and overexpressed the truncated gene ILV2 that codes for acetolactate synthase. The best constructed strain produced more than 20 g of glycerol/L from glucose under micro-aerobic conditions and 16 g of glycerol/L under anaerobic conditions. The increase in glycerol production led to a drop in ethanol and biomass accumulation.