Soil microbial biomass nitrogen (MBN) contains the largest proportion of biologically active nitrogen (N) in soil, and is considered as a crucial participant in soil N cycling. Agronomic management practices such as crop rotation and mono-cropping systems, dramatically affect MBN in agroecosystems. However, the influence of crop rotation and mono-cropping in agroecosystems on MBN remains unclear. A meta-analysis based on 203 published studies was conducted to quantify the effect of crop rotation and mono-cropping systems on MBN under synthetic N fertilizer application. The analysis showed that crop rotation significantly stimulated the response ratio (RR) of MBN to N fertilization and this parameter reached the highest levels in upland-fallow rotations. Upland mono-cropping did not change the RR of MBN to N application, however, the RR of MBN to N application in paddy mono-cropping increased. The difference between crop rotation and mono-cropping systems appeared to be due to the various cropping management scenarios, and the pattern, rate and duration of N addition. Crop rotation systems led to a more positive effect on soil total N (TN) and a smaller reduction in soil pH than mono-cropping systems. The RR of MBN to N application was positively correlated with the RR of mineral N only in crop rotation systems and with the RR of soil pH only in mono-cropping systems. Combining the results of Random Forest (RF) model and structural equation model showed that the predominant driving factors of MBN changes in crop rotation systems were soil mineral N and TN, while in mono-cropping systems the main driving factor was soil pH. Overall, our study indicates that crop rotation can be an effective way to enhance MBN by improving soil N resources, which promote the resistance of MBN to low pH induced by intensive synthetic N fertilizer application.