The alfalfa( Medicaga sativa Gannong No. 3) growing in second year was used to study its nitrogen metabolism and nitrogen accumulation under different nitrogen application levels( 0( CK),51. 75,103. 5 kg·hm- 2) and different mowing times. The results showed that compared with CK,the nitrogen treatments significantly( P 0. 05) increased the activity of nitrate reductase( NR),glutamine synthetase( GS) and transaminase( GOT,GPT),and the contents of soluble protein and total protein in leaves. The activity of tested nitrogen metabolism enzymes reached thehighest when nitrogen application level was 103. 5 kg·hm2. Correlation analysis showed that the relationship between nitrogen application level( x) and total protein content( y) follow a quadratic equation y =- 0. 113 x2+ 53. 632 x + 1 681. 911( R2= 0. 999**). And the nitrogen accumulation positively correlated to nitrogen metabolism activity and soluble protein content( P 0. 05),This suggested that nitrogen fertilization could promote the activity of nitrogen metabolism enzyme.