Accumulation of trehalose, which is a disaccharide composed of 2 molecules of glucose, is generally considered to be a critical determinant in improving the stress tolerance of the baker’s yeast Saccharomyces cerevisiae. To retain this accumulation in yeast cells, we constructed diploid homozygous trehalase (trehalose-hydrolyzing enzyme)-deficient mutants by the gene disruption method form commercial baker’s yeast. During fermentation, degradation of intracellular trehalose was inhibited in the trehalasedeficient mutants. Their freeze tolerance was maintained at a higher level than that of the parent strain. Due to the improved freeze tolerance exhibited by the trehalase-deficient mutants, these strains may be suitable for use in the baking process of frozen dough.