Rice yield and quality are adversely affected by increasing global surface temperature, and are strongly attributed to high night temperature (HNT) than high daytime temperature. However, the molecular mechanism underlying the heat‐tolerant characteristics of rice remains unclear. In the present study, we compared the proteomes of heat‐tolerant and ‐sensitive lines of rice at early milky stage using an iTRAQ method. We have identified 38 differentially expressed proteins between the two lines, of which 32 proteins have been functionally annotated in NCBI and/or the UniProt database. These proteins were then classified into seven functional subgroups, which include signal transduction, transcript regulation, oxidation, defense response, transport, energy metabolism, and biosynthesis. Further analysis indicated that HNT stress could disrupt the redox equilibrium of plant cells, which in turn triggers the calcium‐dependent protein kinase and COP9 signalosome, thereby regulating downstream genes/proteins that are involved in the HNT response. The candidate proteins may provide genetic resources for the improvement of heat‐tolerant characteristics in rice, and the proposed model for signal transduction and transcriptional regulation may facilitate in the elucidation of the molecular mechanism underlying the response to HNT stress in rice.