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Nitrogen (N) fertilizer is a critical element that affects rice yield. However, its effects on dry matter accumulation (DMA), N accumulation, and their physiological mechanisms with grain yield and N utilization efficiency still lack in-depth study. Three large-scale japonica rice varieties—Jinxiangyu 1, Nanjing 46, and Huaidao 5—were used in two field experiments with varying N fertilizer application rates to examine grain yield and N utilization efficiency. The results showed that: (1) In the range of 0~360 kg ha−1 total N application rate (TNAR), the rice yields of the three cultivars were maximum under the TNAR at 270 kg ha−1. The optimal TNAR for the highest yield of Jinxiangyu 1, Nanjing 46, and Huaidao 5 were calculated based on quadratic regressions with values of 305.5 kg ha−1, 307.6 kg ha−1, and 298.0 kg ha−1, and the corresponding yields were 10.3 t ha−1, 10.6 t ha−1 and 10.2 t ha−1, respectively. The N utilization efficiency decreased gradually with the increase in TNAR, and the recovery efficiency decreased from 35.7~38.19% to 29.61~31.59%. (2) The yield was significantly positively correlated with DMA and N accumulation from the heading stage (HD) to the maturity stage (MA). The DMA and N accumulation of HD-MA were significantly positively correlated with leaf photosynthetic rate, non-structural carbohydrate (NSC) accumulation in stems, root oxidation activity, zeatin (Z) + zeatin riboside (ZR) contents in roots, and nitrate reductase (NR) and glutamate synthase (GOGAT) activity in HD. (3) In the range of 0~216 kg ha−1 panicle N application rate (PNAR), the rice yield was maximum under the PNAR at 108 kg ha−1. The optimal PNAR for the highest yield of Jinxiangyu 1 was calculated based on the quadratic regression with values of 139.5 kg ha−1, and the highest yield was 9.72 t ha−1. The leaf photosynthetic rate, NSC accumulation in stems, root oxidation activity, Z + ZR contents in roots, and NR activity in leaves in rice were higher under 108 kg ha−1 PNAR. Excessive application of panicle fertilizer reduced the above physiological indicators and rice yield. The above results showed that optimizing N fertilizer could increase the leaf photosynthetic rate, NSC accumulation in stems, root oxidation activity, Z + ZR contents in roots, and NR activity from HD to MA, which was beneficial to improving DMA and N uptake during HD-MA, thus improving grain yield and N utilization efficiency in rice. [ABSTRACT FROM AUTHOR]