BACKGROUND: Soil- and plant-produced extracellular enzymes drive nutrient cycling in soils and are thought to be regulated supply and demand for carbon (C) and nutrients within the soil. Thus, agriculture management decisions that alter the balance of plant and supplemental nutrients should directly alter extracellular enzyme activities (EEAs), and EEA stoichiometry in predictable ways. We used a 12-year experiment varying three major continuous grain crops (wheat, soybean, and maize) each crossed with mineral fertilizer (WCF, SCF, and MCF, respectively) or not fertilized (WC, SC, and MC, respectively, as controls). In response we measured the phospholipid fatty acids (PLFAs), EEAs and their stoichiometry to examine the changes to soil microbial nutrient demand under the continuous cropping of crops that differed in the input of plant litter and fertilizer. RESULTS: Fertilizer generally decreased soil microbial biomass and enzyme activity compared to non-fertilized soil. According to enzyme stoichiometry, microbial nutrient demand was generally C- and P-limited but not N-limited. The degree of microbial resource limitation, however, differed among the three crops. The enzymatic C:N ratio was significantly lower by 13.3 and 26.8%, but the enzymatic N:P ratio was significantly higher by 9.9 and 42.4%, in MCF than WCF and SCF, respectively. The abundances of arbuscular mycorrhizal fungi and aerobic PLFAs were significantly higher in MCF than WCF and SCF. CONCLUSION: These findings are crucial for characterizing enzymatic activities and their stoichiometries that drive microbial metabolism for understanding soil nutrient cycles and environmental conditions and for optimizing practices of agricultural management. This article is protected by copyright. All rights reserved.