Continuous and pulsed inputs of labile organic carbon (LOC) into soil are common. However, because soil microbial responses to LOC input are rapid, the relative contributions of respiration derived from LOC to total microbial respiration and their influencing factors remain elusive. Furthermore, although numerous studies have explored the priming effect (PE) of soil organic matter (SOM) mineralization induced by LOC addition, few studies have focused on its short-term effects. There are some indications that the response of soil microbes to LOC input depends on the microbial demand of nutrients, especially C and N. Therefore, variations in soil C and N characteristics may further influence microbial response processes to LOC inputs and the decomposition of LOC and SOM, but a comprehensive understanding of this is lacking. To address this gap, 13C-labeled glucose was added to six temperate grassland soils with different C/N ratios (9.98–12.0), which were collected from areas with different grazing exclusion durations and at different soil depths. The microbial respiration was measured at 9-min intervals across a 105-h period. We found that soil microbes responded rapidly to LOC input, and microbial biomass controlled by soil organic C (SOC) and C/N was the most important factor directly influencing the intensity of the microbial response to LOC input. Grazing and deeper soil layers decreased the respiration derived from LOC and their relative contribution to total respiration, mainly attributed to variations in soil C/N and fungal/bacterial ratio (Fu/Ba). LOC addition stimulated SOM decomposition in all soils and increased respiration of SOM by 11.3–92.4 mg C/g SOC, equivalent to 18.7–266.1 % priming. Grazing and increased soil depth resulted in a greater PE and soil C loss, with soil C/N and SOC content being the most important regulators. Overall, this study revealed the important influence of LOC on soil C fluxes and highlighted the important role of SOM quality and quantity in regulating grassland soil C cycling.