Background Grain chalkiness is a highly undesirable quality trait that adversely affects consumer acceptability and lowers the market value. Except for the genotypic variation among different rice cultivars, this chalkiness is readily influenced by application of chemical fertilizers, particularly nitrogen (N) at the late growth stage of rice. However, it is not fully clear on the molecular mechanism underlying the formation of grain chalkiness caused by N fertilization. Results Using OM052 with the chalkiness rate over 90% as material, experiment was conducted with two N fertilizer topdressing treatments at the panicle initiation stage, including 108 kg N ha-1 applied (N+) and without N topdressing to rice as a control (N0). N+ significantly enhanced the area of endosperm chalkiness and the degree of grain chalkiness. The amylose content of rice grains under N+ was lower than that under N0, while the opposite trend was true for N+–induced change in grain protein content. Proteomic analysis found that a total of 198 proteins differentially expressed between N+ and N0, including 9 up-regulated proteins and 189 down-regulated proteins for rice plants imposed to N+. Approximately 31.3% of these differentially expressed proteins (DEPs) involved in N metabolism (protein synthesis, folding, degradation and storage, and amino acid synthesis and catabolism), 21.7% of DEPs belonged to carbohydrate metabolism (glycolysis, tricarboxylic acid cycle, pentose phosphate pathway, fermentation and starch metabolism), and 17.7% of DEPs participated in stress/defense regarding redox homeostasis and removal of aldehydes. Conclusion Multiple metabolic pathways in the developing caryopsis were affected by N topdressing, especially the mitochondrial respiration, sucrose-to-starch metabolism and N metabolism. The insufficient supply of ATP energy as a result of the significantly lowered mitochondrial respiration induced by N+ regime inhibits the sucrose-to-starch metabolism and starch biosynthesis in developing grains, and is strongly responsible for grain chalk formation under N topdressing.