Update publication information. Rainbow trout (Oncorhynchus mykiss) is a typical cold-water fish, the development of rainbow trout aquaculture was severely hampered via the high temperature in summer. Understanding the regulatory mechanism of rainbow trout response to chronic heat stress can provide a theoretical basis for formulating measures to relieve heat stress. In the study, changes in the biochemical parameters revealed that a strong stress response occurred in rainbow trout at 24 °C, the organisms stress defense system was activated, and the immune system was also affected. Proteome of rainbow trout liver tissues under heat stress (24 °C) and control conditions (18 °C) were performed using DIA/SWATH. A total of 390 DEPs were identified by strict threshold (q-value <0.05 and fold changes >1.5), among them 175 were up-regulated and 225 were down-regulated. Some proteins related to HSP, metabolism and immunity were identified. GO analysis showed that some proteins that were highly induced to express at high temperature were involved in the regulation of cell homeostasis, metabolism, adaptive stress and stimulation. KEGG analysis shows that some pathways play an important role in the regulation of heat stress, such as metabolic pathway, protein processing in endoplasmic reticulum pathway, PPAR signaling pathway and complement and coagulation cascades pathway, etc. PPI network analysis shows HSP90b1 and C3 maybe cooperative to protect the integrity of cell membrane function under heat stress. Our finding provide a comprehensive review of protein expression of rainbow trout liver under heat stress, which helps to formulate strategies for rainbow trout to relieve heat stress during high temperature in summer.