Abstract: This paper presents a study about soil nitrogen (N) variations in the grasslands with alfalfas growing for four years, six years, ten years, twelve years, eighteen years and twenty-six years. The study showed that there were consistent change of total N and available N contents with alfalfas growing for different lengths of time in 0–1000 cm soil. Soil total N and available N contents decreased with soil depth, and tended to remain relatively constant in soils deeper than 350cm below soil surface. In 0–200 cm soil, total N and available N contents in 26a was lower than 4a and 6a, but higher than 10a and 12a. In 200–1000 cm soil, there were little difference among different treatments in total N and available N content, which further illustrated that the recovery of soil nitrogen began in upper soil and high consumption of deep-soil nitrogen in the early alfalfa growth made it difficult to recover soil nitrogen in deep soil. There were positive correlation between SOC and total N, available N and C:N. After six years of alfalfa growth, fertilization should be applied to maintain nitrogen balance in alfalfa grassland.