Nitrogen (N) is an important nutrient for plant growth and yield production, and rice grown in paddy soil mainly uses ammonium (NH4+) as its N source. Previous studies have shown that N status is tightly connected to plant defense; however, the roles of NH4+ uptake and assimilation in rice sheath blight disease response have not been studied previously. Here, we analyzed the effects of different N sources on plant defense against Rhizoctonia solani. The results indicated that rice plants grown in N-free conditions had higher resistance to sheath blight than those grown under N conditions. In greater detail, rice plants cultured with glutamine as the sole N source were more susceptible to sheath blight disease compared to the groups using NH4+ and nitrate (NO3–) as sole N sources. N deficiency severely inhibited plant growth; therefore, ammonium transporter 1;2 overexpressors (AMT1;2 OXs) were generated to test their growth and defense ability under low N conditions. AMT1;2 OXs increased N use efficiency and exhibited less susceptible symptoms to R. solani and highly induced the expression of PBZ1 compared to the wild-type controls upon infection of R. solani. Furthermore, the glutamine synthetase 1;1 (GS1;1) mutant (gs1;1) was more susceptible to R. solani infection than the wild-type control, and the genetic combination of AMT1;2 OX and gs1;1 revealed that AMT1;2 OX was less susceptible to R. solani and required GS1;1 activity. In addition, cellular NH4+ content was higher in AMT1;2 OX and gs1;1 plants, indicating that NH4+ was not directly controlling plant defense. In conclusion, the present study showed that the activation of NH4+ uptake and assimilation were required for rice resistance against sheath blight disease.