Nitrate reductase (NR) is an important enzyme for nitrate assimilation in plants, and post-translational phosphorylation regulates NR activity. To evaluate the impact of the dephosphorylation of nitrate reductase 1 (NIA1) protein on NR activity, nitrogen metabolism and plant growth, NIA1 phosphorylation site directed mutant lines (S532D and S532A) and an OsNia1 over-expression line (OE) were constructed, and the phenotype, NIA1 protein and its phosphorylation level, NR activity, nitrate metabolism and reactive oxygen metabolism of the transgenic lines were analysed. Exogenous NIA1 protein was not phosphorylated in S532D and S532A mutant lines, and their NR activities, activity states of NR and assimilation efficiencies of NO3--N were higher than those in Kitaake (WT) and OE. The changes in these physiological and biochemical indexes in the OE line were less than in S532D and S532A compared to WT. These results suggest that the removal of transcriptional level control had little effect on nitrogen metabolism, but the removal of post-translational modification had a profound effect on it. With the removal of NIA1 phosphorylation and the improvement in the nitrate assimilation efficiency, the plant height and chlorophyll content of S532D and S532A decreased and the hydrogen peroxide and malondialdehyde contents of rice seedlings increased, which may be related to the excessive accumulation of nitrite as an intermediate metabolite. These results indicated that the phosphorylation of NR may be a self-protection mechanism of rice. The reduced phosphorylation level of nitrate reductase improved the assimilation of nitrate, and the increased phosphorylation level reduced the accumulation of nitrite and prevented the toxic effects of reactive oxygen species in rice.