Long-term grazing alters soil resource availability and microbial biomass stoichiometry in grassland ecosystems. Exploring the relationship between soil stoichiometric imbalance and microbial carbon use efficiency (CUE) in grazed desert steppe can help understand soil carbon dynamics from a microbial perspective. Based on a long-term grazing platform in Stipa breviflora desert steppe of Inner Mongolia established in 2004, taking heavy, moderate and light grazing intensities, using no grazing as a control, we measured soil available nutrients, microbial biomass and associated enzyme activities for their acquisition, and calculated soil microbial CUE using ecological stoichiometry. The results showed that grazing inhibited soil microbial CUE by 1.0%-10.3%. Soil C:N imbalance was significantly increased by 20.6% in the moderate grazing treatment, while both C:P imbalance and N:P imba-lance were significantly increased by 20.7% and 25.2% in the heavy grazing treatment, indicating that soil microbial communities were more susceptible to N and P limitation. Soil microbial communities maintained their stoichiometric balance by regulating the threshold of elemental stoichiometry ratios and the production of extracellular enzymes. Structural equation modelling (SEM) results indicated that stoichiometric imbalance indirectly affected microbial CUE by altering the threshold of elemental stoichiometry ratios, microbial biomass stoichiometry, and enzyme stoichiometry.