Glutelin, the key storage substance for determining rice quality, was sensitive to warming and nitrogen. However, the relationship between the structural properties of glutelin and rice quality needs to be further investigated under warming and nitrogen. The higher glutelin level was responsible for deteriorating quality under warming and additional nitrogen. The key amino acid components for glutelin were less affected by temperature and nitrogen, whereas glutelin subunit level was sensitive to nitrogen. A lower-ordered sequence for glutelin secondary structure may be involved in deteriorating rice quality for inferior spikelets. The higher level of disulfide bonds may not affect the texture properties of cooked rice. Overall, the results contributed to understanding rice quality formation under warming, as well as a theoretical basis for adjustment of protein extraction process to meet the needs of food processing industry in combination with cultivation measures in light of warming.