To investigate the protective mechanism of yeast hydrolysate (YH) on human umbilical vein endothelial cell injury induced by H₂O₂, antioxidant activity of YH was studied. The results show that the median effective concentration (EC₅₀) of YH on DPPH scavenging is 1.85 mg/mL, showing good antioxidant activity. And YH of high-dose (500 μg/mL) can significantly alleviate the cell proliferation inhibition induced by H₂O₂, reduce malondialdehyde (MDA) production and increase the level of superoxide dismutase (SOD), thereby resisting cell oxidative damage of HUVEC caused by H₂O₂. Moreover, transcriptomics sequencing reveals that YH can significantly up-regulate the expression of genes SOD2 and HMOX1. The research results not only provide basic data for YH to reduce endothelial cell oxidative damage, but also provide a theoretical basis for the application of YH in the fields of antioxidation functional foods and medicine.