Abstract: Field plot and sand pot experiments were carried out to study the effects of four different nitrogen fertilizer levels (0, 50, 100, 150 kg/ha) and two ways of inoculating rhizobia (soaking seeds with rhizobia in autumn and furrow inoculating rhizobia in spring) on the nitrogen fixation ability, growth characteristics and seed yield of alfalfa by using a ¹⁵N natural abundance method. The field experiments were located at the grassland ecological research station in Hutubi County of Xinjiang Agricultural University during the growing seasons 2009 to 2010. The nodule numbers and nodule fresh weight were significantly improved by inoculating rhizobial strain ACCC17544, and attained the maximum percentage and amount of nitrogen fixation (77.8±0.508)% and (452.51±2.568) kg/ha respectively under the lower level of 50 kg/ha nitrogen application. Also, inoculating rhizobia promoted growth, increased above ground dry matter accumulation, number of branches, number of pods and seed yield of alfalfa. The seed yield obtained had an average increase rate of (59.7±0.118)% that was significantly more than those in uninoculated, no nitrogen fertilization and other nitrogen rate treatments. Nitrogen application above 50 kg/ha inhibited nodulation and the percentage and amount of nitrogen fixation were reduced. With increasing amounts of nitrogen fertilizer, the inoculating rhizobial efficiency for improving alfalfa growth were obviously weakened, and the biomass, number of pods, and extent of yield increase were reduced. Soaking seeds with rhizobia in autumn combined with nitrogen application of 50 kg/ha was the best combination for increasing seed yield in the field plot experiments. There was a significant positive relationship between the amount of rhizobial nitrogen fixation and seed yield.