Abstract: Heat is the most common physical agent that denatures proteins. Heat-induced denaturation unfolds protein structure, exposes surface hydrophobic groups and dissociates proteins into their constituent subunits. Denaturation decreases protein solubility, resulting in aggregation of the unfold molecules due to changes in functional properties, such as water absorption, gelation, foaming, and emulsification. An in-depth study was carried out to investigate the physicochemical properties and aggregation characteristics of red kidney bean protein isolate (KPI) during heating and to determine the influence of different temperatures on the thermal aggregation behavior of KPI. The protein solubility, turbidity, content of total and free sulfhydryl groups, subunit composition, and protein secondary structure of native KPI aqueous dispersions (2% g/mL) thermally treated at different temperatures (50, 60, 70, 80, 90 and 95 ℃）were analyzed by different detection methods and techniques. High temperature caused the dissociation and association of protein subunits leading to the formation of soluble and insoluble aggregates, which reduced the solubility of the protein to some extent and increased the turbidity of the protein solution. Results of protein concentration showed that the solubility of red kidney bean protein increased initially and then decreased with the increase of temperature. The solubility of KPI increased by more than 82% at 70 ℃, but decreased to 65.63% at 95 ℃ (near denaturation temperature 93.4 ℃). The turbidity of the protein solution of red kidney bean increased with temperature. Results of SDS-PAGE gel electrophoresis revealed that proteins of red kidney bean tended to form macromolecule aggregates at high temperature. The protein profiles obtained from SDS-PAGE analysis performed in the absence and presence of β-mercaptoethanol were not significantly different when thermal treatment was carried out at a temperature below 90 ℃. A new band with a molecular weight of approximately 135 kDa appeared in the electrophoretic pattern at 90 ℃. The subunit bands with a molecular weight of approximately 90 kDa disappeared in the electrophoretic pattern at 95 ℃, indicating that the protein subunits underwent dissociation and aggregation to a certain extent during heating. A gradual decrease in the content of total sulfhydryl groups was observed and the amount of free sulfhydryl groups initially increased and then decreased as the temperature increased. These results suggested that thermal treatment enhanced protein aggregation and oxidation of SH group. Results of circular dichroism spectroscopy revealed that heat treatment affected the secondary structure of KPI, whereas the content of α-helix in KPI decreased with the rising heating temperature, and the content of random coil increased, but there was no apparent change in the content of β-strand and β-turn. Fluorescence analysis showed that red-shift of KPI solution occurred when the temperature was over 80 ℃, indicating that thermal treatments greatly changed the secondary and tertiary structure of KPI. This implies that high temperature destroys the tertiary structure of KPI and increases the polarity of protein microenvironment. The results can provide the important theoretical basis for controlling the thermal behavior and aggregation properties of red kidney bean products.