Abstract: In order to provide theoretical basis for scientific and reasonable fertilization management in seedling cultivation, studies were made of effects of different application amount of nitrogen fertilizer on the physiological characteristics of 4-years-old Acer truncatum in the field conditions. There were four nitrogen fertilizer application rates: 0 g·plant⁻¹ (CK, Y1), 8 g·plant⁻¹ (Y2), 16 g·plant⁻¹ (Y3) and 24 g·plant⁻¹ (Y4), respectively. The potassium fertilizer application rate was 10 g·plant⁻¹ for all the nitrogen fertilizer treatment. The experiment was conducted in a simple plot design with 3 repetitions. The results showed that 1) from May to September, the chlorophyll content of Y3 increased by 41.19%, 96.20%, 116.89%, 135.20%, 31.92% respectively as compared with the control. And there was no significant difference between Y4 and Y3, and Y3 was significantly higher than Y2. 2) In June, August and September, the flavone content of Y3 increased by 2.98%, 9.36% and 5.01% respectively as compared with Y4, displaying no significant difference. Y3 was significantly higher than Y2 and the control. 3) From May to August, the NR (nitrate reductase) activity of Y3 increased by 40.59%, 35.53%, 37.76%, 46.21% respectively as compared with the control. There was no significant difference between Y4 and Y3. 4) From May to July, soluble protein contents of Y1, Y2 and Y3 were significant higher than those of the control. From August to September, soluble protein content of Y3 was higher than that of the control, increasing by 26.53% and 14.41% respectively. In summary, Y3 treatment was the recommended fertilization treatment in the seedling cultivation of Acer truncatum.