Arid and semi-arid ecosystems significantly influence the carbon cycle, with soil microbes playing a crucial role in this process. However, our understanding of the specific factors influencing nutrient limitation of soil microbial communities and their contribution to carbon sequestration still remains limited, especially in the context of the coupling effect of altitude and slope aspects. Here, we aim to determine soil microbial nutrient limitation and carbon use efficiency (CUE) characteristics and their drivers on different slope aspects along the altitudinal gradient in the Helan Mountains. We determined physicochemical parameters, microbial biomass, and extra-cellular enzyme activities (EEAs) of soil samples collected on different slope aspects (sunny and shady) and altitudes (low, middle, and high). Results displayed that the effect of the slope aspect on soil physicochemical parameters and microbial biomass was notably less significant compared to that of the altitude. Variation in EEAs was accompanied by similar characteristics. Vector lengths were not characterized by significant variation across the altitudinal gradients or between slope aspects, while vector angle exceeded 45 degrees, with higher values observed at high altitudes. Microbial CUE exhibited significant changes on the sunny slope, with the highest value observed at high altitudes. The primary determinants influencing soil microbial nutrient limitation and CUE on the sunny slopes were total nutrients and microbial biomass. In contrast, soil pH, microbial biomass ratios and soil available nutrients played vital roles on the shady slope. This study revealed differences in microbial nutrient limitation status and CUE between slope aspects and their driving factors, providing valuable insights linking microbial resource limitation and nutrient cycling to environmental gradients. We suggested that both altitude and slope aspect be considered essential factors for comprehensive analysis when studying soil microbial nutrient limitation status and CUE in the arid and semi-arid mountainous ecosystem.