Tillage practices can influence soil microbial carbon use efficiency (CUE), which is critical for carbon cycling in terrestrial ecosystems. The effect of tillage practices could also be regulated by nitrogen (N) addition. However, the soil microbial mechanism about N fertilizer effect on microbial CUE under no-tillage is still unclear. We investigated how N fertilizer regulates the effect of tillage management on microbial CUE through changing microbial properties and further assessed the impact of microbial CUE on particulate (POC) and mineral-associated organic matter carbon (MAOC) using a 16-yr field experiment with no-tillage (NT) and conventional tillage (CT), both of which combined with 105 (N1), 180 (N2), and 210 kg N ha-1 (N3) N application. We found that microbial CUE increased with increasing N application rate. NT increased microbial CUE compared with CT under N1. The bacterial and fungal diversities of NT was higher than CT and N application decreased their diversities in the 0-10 cm layer. The partial least squares path model showed that bacteria diversity, fungal diversity, and fungal community structure played more critical roles in increasing microbial CUE. Furthermore, POC and MAOC under NT were higher than CT and they also increased with increasing N application rate. This could be explained by the finding that increasing microbial CUE induced by N application had the potential to increase POC and MAOC. Overall, N addition is an important pathway to influence microbial CUE, which is mainly regulated by bacterial and fungal diversities rather than their biomass under no-tillage.