Grassland converted to cropland affected soil physical and chemical properties and soil microbes. However, these parameters were often studied separately and their combined responses to grassland reclaim remain unclear. To evaluate the impacts of grassland cultivation on soil microbial communities (based on phospholipid fatty acids, PLFAs) and the links between soil microbes and physicochemical properties, we performed a paired field experiment following the conversion from native grasslands to 30-60 year-old cropland in the agro-pastoral ecotone of northern China. The concentrations of soil organic carbon (SOC), soil total nitrogen (STN) and the soil microbial biomass consistently decreased with grassland conversion to cropland. Grassland conversion also significantly decreased the relative abundance of bacterial and fungal PLFAs and the fungal to bacterial ratio (F:B) at 0-10cm soil layer, but those parameters remained unchanged below 10cm soil layers. Grassland conversion affected the microbial biomass mainly through soil C and N content rather than soil pH, moisture and aggregation. These findings revealed that cultivation-induced soil nutrient loss may enhance soil microbe depletion and affect microbial community assembly (shifts in fungi, AMF, Act, GP, and GN bacteria). This implies that conversion of grassland to cropland should be avoided because of the risk of degradation of soil nutrient and microbes.