Abstract: Intercropping is an efficient model for utilizing time and space resources and is a typical diversified agriculture cropping pattern. However, how the diversified planting of aboveground crops affects subsurface microbial diversity and N regulation remains uncertain. Therefore, in this study, the Biolog-EcoMicroPlate cultivation method was used to analyze soil microbial metabolic activity, diversity, and utilization of six carbon sources in potato mono- (MP) and inter-cropping (IP) soils under four N levels (N0, 0 kg∙hm⁻²; N1, 62.5 kg∙hm⁻²; N2, 125 kg∙hm⁻²; and N3, 187.5 kg∙hm⁻²). The results showed that compared with N0 treatment, N application (N1, N2, and N3 treatments) increased the average well color development (AWCD) values by 32.1%–100.2%, Shannon index by 3.3%–7.1%, and Simpson index by 14.8%–19.2%, and the increase peaked at the N1 treatment. Compared to potato monocropping under the same N application rates, potato intercropping with maize increased AWCD values and Shannon and Simpson indices, but there was a statistically significant difference only in the N0 treatment (P<0.05). Moreover, N application significantly affected the microbial metabolic activity of the six carbon sources (P<0.05). N application increased the microbial utilization of the six carbon sources, except for carbohydrates in monocropping soil. N application increased the microbial utilization of polymers, amines, and phenolic compounds (recalcitrant carbon sources), but decreased the utilization of carbohydrates, carboxylic acids, and amino acids (active carbon sources) in intercropping soil. The Mantel test showed that soil temperature, soil water content, soil organic carbon, soil ammonium N, and potato biomass were the main factors affecting the microbial AWCD values and diversity indices; however, ammonium N only had a significant effect on potato intercropping soil (P<0.05). Furthermore, the average clustering coefficient value in monocultured soil (0.930) was slightly higher than that in intercropped soil (0.907), indicating that potato intercropping with maize weakened the stability of soil microbial metabolic processes compared with potato monocropping ecosystem, leading to microbial carbon metabolism being more sensitive to changes in the soil microenvironment and further weakening the promoting effect of intercropping. Overall, diversified planting of aboveground crops increased subsurface microbial metabolic activity and diversity; however, this process was significantly regulated by N application. This indicates that a reasonable aboveground diversity could accelerate the soil carbon cycle and realize the efficient utilization of soil nutrients and sustainable agricultural development.