Grain filling, a crucial stage of grain yield formation in rice, is usually affected by the panicle nitrogen (N) fertilization. Field and pot culture experiments were conducted to explore the underlying mechanisms of N effect. Two rice cultivars with high lodging resistance were grown in the field and pot. Four panicle N fertilization treatments were conducted in 2006 and repeated in 2007. The result showed that medium level of panicle N fertilization treatment (NM) enhanced the accumulation and translocation of non-structural carbohydrate (NSC) in the stem and sheath. Compared with non-nitrogen treatment (N0), NM promoted the translocation of labeled 13C from stem and sheath to grain. But, low level of panicle N fertilization treatment (NL) and high level of panicle N fertilization treatment (NH) showed the negative effect. The endosperm cell, grain length, and grain width of NM increased more quickly than that of N0 from 4 to 10 d after anthesis. During the early period of grain filling, sucrose-phosphate synthase (EC 2.4.1.14, SPS) activity were significantly higher for the NM treatment than those of the NL and NH treatments. Sucrose synthase (EC 2.4.1.13, SuSase) activity in the grains was substantially enhanced by NM, with the duration of higher activity being longer than those of the other treatments. At maturing stage, NM significantly increased the filled grain number, the seed-setting rate, and the grain weight compared with NL and NH. The results suggest that NM have a positive effect on the activities of enzymes of physiological importance, thereby increasing the grain size and promoting grain filling. Grain filling, a crucial stage of grain yield formation in rice, is usually affected by the panicle nitrogen (N) fertilization. Field and pot culture experiments were conducted to explore the underlying mechanisms of N effect. Two rice cultivars with high lodging resistance were grown in the field and pot. Four panicle N fertilization treatments were conducted in 2006 and repeated in 2007. The result showed that medium level of panicle N fertilization treatment (NM) enhanced the accumulation and translocation of non-structural carbohydrate (NSC) in the stem Compared with non-nitrogen treatment (N0), NM promoted the translocation of labeled 13C from stem and sheath to grain. But, low level of panicle N fertilization treatment (NL) and high level of panicle N fertilization treatment (NH) showed the negative effect. The endosperm cell, grain length, and grain width of NM increased more quickly than that of N0 from 4 to 10 d after anthesis. During the early period of grain fill Sucrose synthase (EC 2.4.1.13, SPS) activity were significantly higher for the NM treatment than those of the NL and NH treatments. Sucrose synthase (EC 2.4.1.13, SuSase) activity in the grains was substantially enhanced by NM , with the duration of higher activity being longer than those of the other treatments. At maturing stage, NM significantly increased the filled grain number, the seed-setting rate, and the grain weight compared with NL and NH. The results suggest that NM have a positive effect on the activities of enzymes of physiological importance, thereby increasing the grain size and promoting grain filling.