Cross protection is a widely existed phenomenon in microorganisms which subjected to a mild stress develop tolerance to other stresses, yet the underlying mechanisms for this protection have not been fully elucidated. Here, we report that heat preadaptation induced cross protection against acid stress in Tetragenococcus halophilus, and the cross protective mechanisms were revealed based on cell surface characterizations and transcriptomic analysis. The results showed that heat preadaptation of T. halophilus at 45 °C for 1.5 h improved the acid tolerance of cells at pH 2.5, and the preadapted cells exhibited higher pHi compared with the un-preadapted cells during acid stress. Analysis of the cell surface properties suggested that the heat-treated cells displayed smoother surface, lower roughness and higher integrity than those of untreated cells. Meanwhile, the distributions of membrane fatty acids also changed in response to acid stress, and the treated cells reveled lower ratio of unsaturated to saturated fatty acids. RNA-Sequencing was employed to further elucidate the cross protective mechanism induced by heat preadaptation, and the results showed that the differentially expressed genes (DGEs) were mainly involved in cellular metabolism and membrane transport during heat preadaptation. A detailed analysis of gene expression profile of cells between heat treated and untreated revealed that genes associated with energy metabolism, amino acid metabolism and genetic information processing were induced upon heat stress. Results presented in this study may broaden our understanding on cross protection and provide a potential strategy to enhance the performance of cells during industrial processes.