The anatomical structure of leaf conducting tissue and photosynthetic characteristics of alfalfa (Medicago sativa L.) were studied based on two nitrogen forms (nitrate nitrogen and ammonium nitrogen) and five nitrogen levels (0,105,210,315,420 mg·L^-1).Two cultivars,‘Gannong No.3’ and ‘Longdong’,were selected as materials,and the experiment was conducted outdoors (in flashing net room) using sand culture method.The results showed that:(1) Compared with non-nitrogen treatment,the anatomical structure of leaf conducting tissue of both alfalfa cultivars changed significantly under both nitrogen treatments.The vascular bundle area,xylem area,phloem area and vessel numbers of leaf conducting tissue increased significantly.The photosynthetic rate,stomatal conductance,leaf area and chlorophyll content also increased significantly (P<0.05),and the maximum number occurred at nitrogen level 210 mg·L^-1.(2) Compared with NO₃^--N treatment,the vascular bundle area,xylem area,phloem area,vessel number,photosynthetic rate,stomatal conductance,leaf area and chlorophyll content increased more under NH₄⁺-N treatment.(3) Compared with cultivar ‘Longdong’,the vascular bundle area,xylem area,phloem area,vessel number,photosynthetic rate,stomatal conductance,leaf area and chlorophyll content performed better in cultivar ‘Gannong No.3’.The data showed that nitrogen can improve the anatomical structure of leaf conducting tissue and photosynthetic characteristics of alfalfa by increasing photosynthesis.The combination of NH₄⁺-N and 210 mg·L^-1 performed best in all treatments.The anatomical structure of leaf conducting tissue was improved,the vascular bundle was the biggest,both phloem and xylem showed largest area and developed well,and the number of vessel was also the largest;and ‘Gannong No.3’ performed better than ‘Longdong’.