Abstract: Soil moisture significantly affects crop response to fertilizer application. This study investigated the effect of nitrogen application on the growth of alfalfa (Medicago sativa) in the middle and lower reaches of the Yangtze River and evaluated the interaction between soil moisture and nitrogen application rate effects with a semi-controlled pot experiment conducted in Nanjing. The experiment included three soil moisture levels (50%, 70% and 90% of maximum water holding capacity) and four nitrogen application rates (0, 90, 180, 270 kg/ha). Dry matter accumulation and nitrogen use efficiency increased with increasing soil moisture level. Under the same soil moisture level, dry matter accumulation and nitrogen use efficiency increased with appropriate N application rates, but decreased with excess nitrogen application. The interaction effect between soil moisture and nitrogen application rate was statistically significant. The effect of fertilizer nitrogen was significantly enhanced at higher soil moisture levels. With increased soil moisture, the effect of nitrogen application at moderate rates was also enhanced. Therefore, when soil moisture level is lower, nitrogen application should be carried out with irrigation. In the middle and lower reaches of the Yangtze River where there is high soil moisture, more nitrogen application could increase forage yield and nitrogen use efficiency of alfalfa.