It remains unclear whether microbial carbon limitation exists in the rhizosphere, a microbial hotspot characterized by intensive labile carbon input. Here, we collected rhizosphere soils attached to absorptive and transport roots and bulk soils in three alpine coniferous forests and evaluated the limiting resources of microbes based on the responses of microbial growth (18O incorporation into DNA) and respiration to full-factorial amendments of carbon, nitrogen, and phosphorus. The results showed that adding carbon enhanced microbial growth and respiration rates in the rhizosphere soils by 1.2- and 10.3-fold, respectively, indicating the existence of carbon limitation for both anabolic and catabolic processes. In contrast, the promoting effects of nutrient addition were weak or manifested only after the alleviation of carbon limitation, suggesting that nutrients were co-limiting or secondarily limiting resources. Moreover, the category and extent of microbial resource limitations were comparable between the rhizosphere of absorptive and transport roots, and between the rhizosphere and bulk soils. Overall, our findings offer direct evidence for the presence of microbial carbon limitation in the rhizosphere.