AimsThe restoration of degraded grassland ecosystems significantly impacts microbial carbon use efficiency (CUE) by altering ecoenzymatic stoichiometry and soil nutrient availability. However, the spatiotemporal variability in resource limitations and the primary factors regulating microbial CUE across ecosystems being restored under different environmental conditions (arid versus mesic) remain unclear.MethodsWe measured the activities of carbon (C)-, nitrogen (N)-, and phosphorus (P)-acquiring enzymes, utilized an extracellular enzyme stoichiometry model to evaluate microbial resource limitations, and investigated the drivers of microbial CUE over a 20-year restoration chronosequence in arid and humid regions.ResultsSoil organic carbon (SOC) increased by 96.33% (from A0 to A20) in arid grasslands and 79.86% (from H0 to H20) in humid grasslands, with SOC strongly coupled to microbial biomass carbon. Microbial CUE was higher in arid regions (median = 0.17; IQR: 0.16-0.21) compared to humid (median = 0.12; IQR: 0.11-0.13) regions, suggesting a greater potential for soil C sequestration in arid regions. Soil microbial communities in both regions were co-limited by C and N. However, arid grasslands exhibited a progressive alleviation of microbial C limitation and exacerbated N limitation. Microbial CUE exhibits a significantly negative correlation with C limitation, highlighting the interplay between C dynamics and microbial metabolic activities during vegetation restoration.ConclusionsNutrient imbalance and resource limitations significantly impact microbial CUE across both regions, with soil water content being a particular factor in arid regions. Targeted management strategies for managing resource limitations in different ecosystems during vegetation restoration could enhance CUE and promote soil C sequestration.