Nitrogen (N) fertilization has been recognized as improving the grain beneficial micronutrients, including Zn and Fe, in rice. However, only a few studies have explicitly focused on N-induced variation in anti-nutritional components such as phytic acid (PA), PA synthesis-related gene expression, and variation in grain protein fractions. Therefore, in this study, two culture systems (hydroponic and detached panicle culture systems) were used to elucidate the influence of N application on PA concentration and its relation to the grain protein fractions, such as albumin, globulin, prolamin, and glutelin, and total protein in rice. Results showed that N application generally decreased the grain PA concentration in brown rice and down-regulated the PA synthesis-related genes in the lipid-independent pathway. In contrast, total grain protein and its fractions concentrations increased significantly. For grain positional distribution, PA and protein concentration were generally higher in the aleurone fraction than in the milled rice, regardless of N application. However, higher N application decreased the PA in both aleurone fraction and milled rice, while increased the grain protein fractions mainly in milled rice. These findings imply that N application could substantially improve the rice nutrition by reducing the PA while increasing the protein concentration. Hence, these findings may provide critical bases for rice nutritional improvement through optimal N management.