A 3 a field experiment, consisting of 6 croppings, was conducted to investigate effects of N fertilization on content and fractionation of soil organic N relative to N application rate and crop rotation mode, paddy/upland (rice/rapeseed, RR) rotation and upland (cotton/rapeseed, CR) rotation. Results show that after the 3 years of crop rotations cultivations, soil total N content in the 0~20 cm soil layer increased remarkably in plots more than 300 kg hm^-2 in N input. Compared with CK (No N fertilization, N0-0), soil total N content increased by 13.6%~23.5% and 15.0%~23.0%, respectively, in the treatments 300 kg hm^-2 and 375 kg hm^-2 in N application rate under either RR or CR rotation. The increase in soil total acidolysable N content accounted for most of the increase in soil total N. Although no significant difference in soil total acidolysable N content was observed between plots under RR and CR rotations, fractionation of the soil acidolysable N varied between plots. The proportion of soil acidolysable ammonium N increased by 33.8% in the plots under RR rotation, much lower than that (53.9%) in the plots under CR rotation, but the increment of unidentified soil acidolysable N in proportion was higher in plots under RR rotation (36.0%) than that (16.6%) in the plots under CR rotation. To sum up, reasonable N fertilization may significantly increase soil organic N content. Fractionation of the soil organic N pool varies sharply between plots under RR and CR rotations. It is, therefore, of important significance to optimize N fertilization for high crop yield and high N utilization efficiency in the light of the characteristics of soil organic N transformation under different rotation systems.