Aims <p>Understanding changes in soil microbial carbon use efficiency (CUE) and microbial biomass turnover time are of great significance for increasing ecosystem C sequestration and achieving the strategic goal of C neutrality. However, the response of soil microbial CUE to nitrogen (N) deposition during extreme drought and wetness events in saline–alkaline grasslands remains unclear.</p> Methods <p>Herein, using an innovative substrate-independent method by incorporating the isotope from H₂¹⁸O into the DNA of microorganisms, the soil microbial CUE under N addition and precipitation changes (± 50% of natural precipitation) were studied.</p> Results <p>Results showed that the microbial CUE was ranged from 0.29 to 0.59, with an average of 0.46. N addition and precipitation changes alone had no significant effect on soil microbial CUE. N addition combined with drought showed significantly negative influences on the relative changes in soil microbial CUE (decreased by 16.3%), while showed significantly positive influences on microbial biomass turnover time (increased by 96.0%). There was a negative correlation between the soil microbial CUE and microbial biomass turnover time. Microbial CUE was positively correlated with plant biomass, soil water content, and soil bacterial abundance, whereas it was negatively correlated with soil dissolved organic C and soil fungal abundance. Variation partitioning analysis showed that plants, soil environment, soil available nutrients, and soil microbes co-regulated the variation in soil microbial CUE, and the plant biomass, soil fungi, and SWC were the core factors that affected soil microbial CUE.</p> Conclusions <p>Overall, we highlighted that N addition reduces soil C sequestration potential under droughts conditions.</p>