The objective of this study was to investigate the protective effects of oat β-glucan (OβG) on yeast subjected to freeze-thaw cycle-induced stress. A range of analytical techniques were employed to identify the underlying molecular mechanisms, including flow cytometry, gas chromatography-mass spectrometry, and quantitative real-time PCR. Following three freeze-thaw cycles, the survival rate of yeast that had been supplemented with 0.5 % OβG was found to be significantly higher than that of the control sample, increasing from 36.21 % to 56.81 %. The addition of 0.5 % OβG resulted in a remarkable reduction in apoptosis, an improvement in cell membrane integrity, and an increase in superoxide dismutase, catalase activity and glutathione content compared to the control group. Furthermore, a noticeable increment in the intracellular trehalose content was observed, from 4.10 mg/g to 7.48 mg/g. OβG modulated the expression of trehalose metabolism-related genes (ATH1, NTH1, NTH2) throughout the freeze-thaw cycle. Therefore, it could be concluded that OβG protected yeast cells against excessive reactive oxygen species and minimised oxidative damage to cellular membranes by upregulating antioxidant enzyme activity and total antioxidant capacity. Moreover, the supplementation of OβG was also found to be effective in increasing intracellular trehalose levels, thereby enhancing the freezing resistance of yeast cells.