Zygosaccharomyces rouxii plays important roles in the brewing process of fermented foods such as soy sauce, where salt stress is a frequently encountered condition. In this study, effect of heat preadaptation on salt tolerance of Z. rouxii and the protective mechanisms underlying heat preadaptation were investigated based on physiological and transcriptomic analyses. Results showed that cells subjected to heat preadaptation (37 degrees C, 90 min) prior to salt stress aroused many physiological responses, including maintaining cell surface smooth and intracellular pH level, increasing Na+/K+-ATPase activity. Cells subjected to heat preadaptation increased the amounts of unsaturated fatty acids (palmitoleic C16:1, oleic C18:1, linoleic C18:2) and decreased the amounts of saturated fatty acids (palmitic C16:0, stearic C18:0) which caused the unsaturation degree (unsaturated/saturated = U/S ratio) increased by 2.4 times when compared with cells without preadaptation under salt stress. Besides, salt stress led to increase in contents of 5 amino acids (valine, proline, threonine, glycine, and tyrosine) and decrease of 2 amino acids (serine and lysine). When comparing the cells pre-exposed to heat preadaptation followed by challenged with salt stress and the cells without preadaptation under salt stress, the serine, threonine, and lysine contents increased significantly. RNA sequencing revealed that the metabolic level of glycolysis by Z. rouxii was weakened, while the metabolic levels of the pentose phosphate pathway and the riboflavin were enhanced in cells during heat preadaptation. Results presented in this study may contribute to understand the bases of adaptive responses in Z. rouxii and rationalize its exploitation in industrial processes. Key points Heat preadaptation can improve high salinity tolerance of Z. rouxii. Combined physiological and transcriptomic analyses of heat preadaptation mechanisms. Provide theoretical support for the application of Z. rouxii.