The carbon (C) use efficiency (CUE) of microorganisms, as important engine of the soil C cycle, determines the proportion of microbial metabolic C allocated to biomass and their C sequestration potential. The continuous input of high-quality C sources in the soil has formed microbial hotspots with higher microbial metabolic activity (e.g., rhizosphere), but it is unclear whether the microorganisms here have a higher microbial CUE, especially lacking verification at the regional scale. Herein, we collected paired rhizosphere and bulk soil samples from 46 alpine coniferous forest sites on the eastern Tibetan Plateau to quantify the difference in microbial CUE between the two soil compartments using the ¹⁸O stable isotope-based techniques. We also determined soil resource availability (i.e., dissolved organic C and nutrient availability) and microbial physiological traits to elucidate the mechanism affecting microbial CUE. The results revealed that the average microbial CUE in the rhizosphere was higher by 103.4% greater than that in the bulk soil, suggesting that the rhizosphere has a greater potential for microbial C sequestration. This is attributed to the differences in soil resource availability between the rhizosphere and bulk soils. Specifically, the rhizosphere has greater microbial biomass C, faster growth and turnover rates, and lower biomass-specific enzyme activity relative to bulk soils, suggesting that microorganisms in the rhizosphere allocate more C to anabolic processes rather than resource acquisition, thereby yielding higher microbial CUE. Collectively, our findings verify the existence of higher microbial CUE in soil hotspots from a rhizosphere perspective and highlight the ecological importance of rhizosphere microbial C anabolism in regulating soil C dynamics.