Background and aims <p>Microbes are the primary decomposers of litter, yet how above and belowground litter inputs contribute to soil microbial structure and function remains unclear across global environmental gradients.</p> Methods <p>Here, we conducted a global meta-analysis including 1,509 observations to explore the global patterns of how leaf addition, leaf removal, root removal, and both leaf and root removal affect structure and function of the soil microbiome as well as the potential mechanism.</p> Results <p>Leaf addition significantly increased soil total microbial biomass and soil respiration by 7.4% and 25.1% respectively. However, the removal of leaves, roots, or both negatively affected total microbial biomass, reducing it by 10.0%, 23.5%, and 17.1%, respectively, and decreased soil respiration by 22.4%, 7.2%, and 33.7%, respectively. Root removal had a more pronounced negative impact on fungal biomass (-23.0%) than on bacterial biomass (-9.3%). The response of soil microbes to litter inputs varied across climates, ecosystem types and duration. Microbial biomass was negatively correlated with mean annual temperature, with stronger effects observed in grasslands than in forests. Also, the most pronounced negative effects of root removal observed in long-term experiments.</p> Conclusions <p>Our work highlighted the distinct roles of above and belowground litter inputs in shaping soil microbial structure and function across global biomes, which are fundamental for predicting the future of terrestrial ecosystems and improving ecosystem management under global change.</p>