Abstract: Frequent mowing and nitrogen enrichment can lead to the degradation of grassland ecosystem. It remains largely unknown that how the soil microbial characteristics, important bio-indicators of soil quality, respond to mowing and nitrogen enrichment. In this study, using a field experiment established in the meadow steppe in Hulunber, Inner Mongolia, we explored the responses of soil properties, microbial biomass, soil respiration, and soil enzyme activities to mowing and nitrogen addition during growing seasons. Mowing significantly reduced microbial biomass carbon, nitrogen and phosphorus, and soil respiration (basal respiration and substrate induced respiration), which might be caused by the moisture- and carbon-limitation. Mowing significantly reduced activities of the enzymes involved in nitrogen acquisition (N-acetyl-β-D-glucosaminidase) and phosphorus acquisition (acidic phosphomonoesterases), which supports the resource allocation theory. Soil pH was significantly reduced by N addition. However, microbial biomass showed no significant response to nitrogen input, implying that soil acidification induced by nitrogen inputs was not profound enough to affect microbial biomass. Nitrogen addition did not affect soil respiration and microbial enzymatic activities, inconsistent with results from most of previous studies conducted in typical steppe. Combination of mowing and nitrogen addition reduced the activity of acidic phosphomonoesterases, which might be due to the increased phosphorus availability under the combined treatment. Combination of mowing and nitrogen addition reduced microbial biomass phosphorus, but increased soil available phosphorus, corresponding to the lowered activity of acidic phosphomonoesterases under the combined treatment. Microbial biomass carbon, nitrogen and phosphorus, and soil respiration peaked in July, which was associated with the high temperature and precipitation in summer. Soil enzymatic activities were higher in the spring and summer than in the late growing season. In summary, our results indicated that mowing would result in the imbalance of soil nutrients and intensify the risk of grassland degradation. In contrary, nitrogen addition exerted no effects on microbial biomass and activity.