Intercropping is an effective cultivation practice to develop sustainable agroecosystems and increase soil organic carbon (SOC) and nitrogen (N) with rich crop diversity. With increased duration, the changes in soil N availability might affect microbial and plant growth and consequently regulate the intercropping effects on SOC sequestration. However, the response of intercropping effects on SOC components to soil N availability has been poorly explored. In this study, we constructed a global database with 939 paired data (intercropping vs. monoculture) from 60 publications. We found that intercropping increased SOC (by 7.94%), microbial biomass carbon (MBC, by 23.73%), particulate organic carbon (POC, by 23.27%), and dissolved organic carbon (DOC, by 16.46%) compared to monoculture. The intercropping effects (percentage change, %) on SOC content strongly depends on soil N availability (soil C: N) and the interactions with duration and crop types. In N-limited soils (soil C: N > 15), intercropping increased SOC and POC contents by 14% and 31%, respectively, which were 2–3 times higher than those in N-rich soils (soil C: N < 15). It implies higher intercropping effects in more N-limited soils. In addition, intercropping increased MBC:SOC by 63% in N-limited but not in N-rich soils, indicating greater contributions of microbial-derived C to SOC storage in N-limited but not in N-rich soils. This might be attributed to that the stimulated microbial decomposition of soil organic matter counterbalanced the microbial-derived C accumulation in N-rich soils. Taken together, these results indicate that soil N availability regulates the intercropping effects on SOC content and the contributions of microbial-derived C to SOC storage. Overall, these findings highlight the importance of soil N availability on SOC content with intercropping, implying that intercropping is a suitable agricultural approach especially in N-limited soils and nutrient management should be carefully considered in long-term intercropping experiments.