Freeze-thaw stress causes various types of cellular damage, survival and/or proliferation defects, and metabolic alterations. However, the mechanisms underlying how cells cope with freeze-thaw stress are poorly understood. Here, model dough fermentations using two baker's yeast strains, 45 and YF, of Saccharomyces cerevisiae were compared after 2 weeks of cell preservation in a refrigerator or freezer. YF exhibited slow fermentation after exposure to freeze-thaw stress due to low cell viability. A DNA microarray analysis of the YF cells during fermentation revealed that the genes involved in oxidative phosphorylation were relatively strongly expressed, suggesting a decrease in the glycolytic capacity. Furthermore, we found that mRNA levels of the genes that encode the components of the proteasome complex were commonly low, and ubiquitinated proteins were accumulated by freeze-thaw stress in the YF strain. In the cells with a laboratory strain background, treatment with the proteasome inhibitor MG132 or the deletion of each transcriptional activator gene for the proteasome genes ( RPN4 , PDR1 , or PDR3 ) led to marked impairment of model dough fermentation using the frozen cells. Based on these data, proteasomal degradation of freeze-thaw-damaged proteins may guarantee high cell viability and fermentation performance. We also found that the freeze-thaw stress-sensitive YF strain was heterozygous at the PDR3 locus, and one of the alleles (A148T/A229V/H336R/L541P) was shown to possess a dominant negative phenotype of slow fermentation. Removal of such responsible mutations could improve the freeze-thaw stress tolerance and the fermentation performance of baker's yeast strains, as well as other industrial S. cerevisiae strains. IMPORTANCE The development of freezing technology has enabled the long-term preservation and long-distance transport of foods and other agricultural products. Fresh yeast, however, is usually not frozen because the fermentation performance and/or the viability of individual cells is severely affected after thawing. Here, we demonstrate that proteasomal degradation of ubiquitinated proteins is an essential process in the freeze-thaw stress responses of S. cerevisiae . Upstream transcriptional activator genes for the proteasome components are responsible for the fermentation performance after freezing preservation. Thus, this study provides a potential linkage between freeze-thaw stress inputs and the transcriptional regulatory network that might be functionally conserved in higher eukaryotes. Elucidation of the molecular targets of freeze-thaw stress will contribute to advances in cryobiology, such as freezing preservation of human cells, tissues, and embryos for medical purposes and breeding of industrial microorganisms and agricultural crops that adapt well to low temperatures.