To investigate the molecular mechanism for the effect of oat β-glucan (OβG) at different concentrations (0.5% and 1%) on freeze tolerance in yeast, yeast cells alone or under the protection of OβG were frozen and thawed up to five times. High-throughput sequencing and bioinformatics were used to elucidate the metabolic pathways and key genes associated with yeast freeze tolerance at the transcriptome level. The results indicated that after three cycles of freezing and thawing, the survival rates of yeast cells supplemented with 0.5% OβG and 1% OβG were higher than that of the control group by 20.60% and 17.08%, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of differentially expressed genes (DEGs) at the transcriptomic level showed that several metabolic pathways including the amino acid metabolism, carbohydrate metabolism, and lipid metabolism pathways played significant roles in the enhancing effect of OβG supplementation on yeast freeze tolerance. Comprehensive analysis revealed that following OβG addition, yeast down-regulated the expression levels of genes related to amino acid metabolism, thereby reducing the consumption of amino acids, while lowering the expression of the VB₆ biosynthesis genes, thereby maintaining intracellular amino acids at a relatively stable level, and consequently contributing to increased survival rates. Meanwhile, adding OβG resulted in significant up-regulation of the trehalose synthase gene in yeast cells and significant down-regulation of the trehalose hydrolase gene, facilitating the accumulation of intracellular trehalose. Additionally, the molecular chaperone genes CNS1 and HSP82, as well as the fatty acid synthesis-related genes FAS1 and PHS1, played crucial roles in the enhancing effect of OβG on yeast freeze tolerance. In summary, OβG has the potential to enhance freeze tolerance in yeast under freeze-thaw conditions by regulating multiple metabolic pathways, making it a highly promising cryoprotectant for yeast.