De-oiled rice bran is a good source of high-quality protein; however, the current practice of desolventization at high temperature (110–120 °C) denatures the protein, making its extraction difficult and uneconomical. The present study aims to investigate the effect of low temperature desolventization of de-oiled rice bran (LTDRB) on extraction, yield, and purity of protein and its comparison with protein obtained from high temperature desolventized de-oiled rice bran (HTDRB). The optimal conditions for preparation of protein from LTDRB were: extraction pH 11.00, extraction duration 52 min, and extraction temperature 58 °C resulting in an extraction efficiency, yield, and purity of 54.0, 7.23, and 78.70%, respectively. The LTDRB showed a positive impact on the color, solubility, foaming capacity and stability of protein whereas the absorption and emulsification properties were better for HTDRB protein. Significant decrease in enthalpy (ΔH) for denaturation was observed for LTDRB protein as compared to HTDRB protein. Scanning electron microscopy analysis revealed that HTDRB protein was more compact than LTDRB protein. LTDRB protein had smaller particle size distribution than HTDRB. Study suggested that low temperature desolventization can result in higher protein extraction with better physico-chemical, structural, and functional properties of protein obtained from DRB.