Mowing is a common practice in grassland management, impacting root biomass, soil properties, and microorganisms by removing aboveground biomass. However, the effects of mowing on these factors have not been thoroughly reviewed. In this study, we evaluated the overall impact of mowing on root biomass, soil properties, microbial biomass, and diversity grasslands by synthesizing 1103 field observations from 103 global publications and assessed whether the effects of mowing varied with grassland type, mowing duration, and stubble height. Results revealed that mowing duration significantly influenced root biomass, transitioning from positive to negative effects with increased duration. Moreover, the effect of mowing on inorganic nitrogen (IN) differed among grassland types, decreasing in semi-humid/humid grasslands but remaining unchanged in arid/semi-arid ones. Stubble height impacted ammonium nitrogen (NH4+-N), decreasing under medium and low stubble height but not under high stubble height. The effect of mowing on microbial biomass nitrogen (MBN) varied with grassland type and stubble height. MBN decreased in semi-humid/humid grasslands and under high stubble height but remained unchanged in arid/semi-arid grasslands or under medium and low stubble height. Additionally, mowing did not change the total microbial biomass, but decreased the biomass of gram-positive bacteria, gram-negative bacteria, and arbuscular mycorrhizal fungi, as well as the bacterial Shannon diversity index, and increased the fungal Simpson diversity index. Overall, this meta-analysis provides insights into how grassland ecosystems respond to mowing, particularly in terms of root biomass, soil properties, microbial biomass, and diversity. It also highlights the necessity for site-specific grassland management strategies.