Laboratory and in situ experiments were done to investigate the influences of cultivation on temperate semi-arid grassland (for 17years spring wheat planted once every two years without fertilization) on soil N2O emission and quantitative variations of related soil microbes.In the laboratory (25℃ and soil moisture 18% ), cultivation increased soil transformations of fertilizer nitrogen ( 100 μg N/g as NaNO3, urea,or as urea with dicyandiamide I μg N/g). The N2O emissions from the cultivated and uncultivated soils with or without nitrogen additions were relatively Iow, and mainly originated from the nitrification. The soil N2O emission due to cultivation decreased somewhat upon no fertilization or NaNO3 addition, but significantly upon urea addition. The role of dicyandiamide as nitrification inhibitor was only considerable in the cultivated soil, and had small influence on decreasing N2O emission in the two soils. The influence of cultivation on soil N2O emission was also reflected by the number variations of microbes related with soil nitrogen transformation in the two soils. Compared to the uncultivated grassland, in situ ammonifiem and denitrifiers in the cultivated grassland quantitatively averagely increased, and aerobic no-symbiotic azotobacters were quantitatively similar, leading to the continued decrease of organic matter content and the decrease of N2O emission from the cultivated grassland soil.