The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD(+)-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1 Delta and gpd2 Delta were studied under alcoholic fermentation conditions. gpd1 Delta mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2 Delta mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1 Delta) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1 Delta) but a slight decrease in the strain ANG2 (gpd2 Delta). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1 Delta) and ANG2 (gpd2 Delta) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.