Heat-induced chalkiness of rice grains is a major concern for rice production, particularly with respect to climate change. Although the formation of chalkiness in the endosperm is suppressed by nitrogen, little is known about the cell-specific dynamics of this process. Here, using picolitre pressure-probe electrospray-ionization mass spectrometry together with transmission electron microscopy and turgor measurements, we examine heat-induced chalkiness in single endosperm cells of intact rice seeds produced under controlled environmental conditions. Exposure to heat stress decreased turgor pressure and increased the cytosolic accumulation of sugars, glutathione, and amino acids, particularly cysteine. Heat stress also led to a significant enlargement of the protein storage vacuoles but with little accumulation of storage proteins. Crucially, this heat-induced partial arrest of amyloplast development led to formation of chalkiness. Whilst increased nitrogen availability also resulted in increased accumulation of amino acids, there was no decrease in turgor pressure. The heat-induced accumulation of cysteine and glutathione was much less marked in the presence of nitrogen, and storage proteins were produced without chalkiness. These data provide important information on the cell dynamics of heat acclimation that underpin the formation of chalkiness in the rice endosperm. We conclude that rice seeds employ multiple strategies to mitigate the adverse effects of heat stress in a manner that is dependent on nitrogen availability, and that the regulation of protein synthesis may play a crucial role in optimizing organelle compartmentation during heat adaption.