The influences of three wheat gluten peptides (WGP-LL, WGP-LML, and WGP-LLL) on the osmotic stress tolerance and membrane lipid component in brewer's yeast were investigated. The results demonstrated that the growth and survival of yeast under osmotic stress were enhanced by WGP supplementation. The addition of WGP upregulated the expressions of OLE1 (encoded the delta-9 fatty acid desaturase) and ERG1 (encoded squalene epoxidase) genes under osmotic stress. At the same time, WGP addition enhanced palmitoleic acid (C16:1) content, unsaturated fatty acids/saturated fatty acids ratio, and the amount of ergosterol in yeast cells under osmotic stress. Furthermore, yeast cells in WGP-LL and WGP-LLL groups were more resistant to osmotic stress. WGP-LL and WGP-LLL addition caused 25.08% and 27.02% increase in membrane fluidity, 22.36% and 29.54% reduction in membrane permeability, 18.38% and 14.26% rise in membrane integrity in yeast cells, respectively. In addition, scanning electron microscopy analysis revealed that the addition of WGP was capable of maintaining yeast cell morphology and reducing cell membrane damage under osmotic stress. Thus, alteration of membrane lipid component by WGP was an effective approach for increasing the growth and survival of yeast cells under osmotic stress. Key points: •WGP addition enhanced cell growth and survival of yeast under osmotic stress. •WGP addition increased unsaturated fatty acids and ergosterol contents in yeast. •WGP supplementation improved membrane homeostasis in yeast at osmotic stress. [ABSTRACT FROM AUTHOR]