The turnover of SOC in soils is strongly influenced by the availability of substrate and nutrients, especially nitrogen (N) and phosphorus (P). Here, we assessed how long-term fertilization modified SOM mineralization in response to added substrate in a tropical forest. We carried out a 90-day incubation study in which we added two structurally similar compounds which differed in microbial metabolic availability: corn cellulose or corn starch to soils collected from a long-term (11 years) factorial N and P fertilization experiment site in a tropical forest in south China. We measured total soil mineralization rate (CO2 efflux) to characterize SOM mineralization and using 13C isotope signatures to determine the source of the CO2 (original soil C or added substrate) and assessed changes in extracellular enzyme activities: acid phosphomonoesterase (AP), β-1,4-glucosidase (BG), β-1,4- N-acetaminophen glucosidase (NAG), phenol oxidase (PHO) and peroxidase (PER), and microbial biomarkers to determine whether nutrient stoichiometry and decomposer communities explain differences in SOM mineralization rates. Total C mineralization increased substantially with substrate addition, particularly cellulose (5.38, 7.13, 5.58 and 5.37 times for N, P, NP fertilization and CK, respectively) compared to no substrate addition, and original soil C mineralization was further enhanced in long-term N (3.40% and 5.18% for cellulose and starch addition, respectively) or NP (35.11% for cellulose addition) fertilized soils compared to control treatment. Enzyme activities were stimulated by the addition of both substrates but suppressed by P-fertilization. Addition of both substrates increased microbial investment in P-acquisition, but only starch addition promoted C investment in N-acquisition. Finally, fungal abundance increased with substrate addition to a greater extent than bacterial abundance, particularly in cellulose-amended soils, and the effect was amplified by long-term fertilization. Our findings indicate that SOM mineralization might be enhanced in N and P enrichment ecosystems, since the litter input can liberate microbes from C limitation and stimulate SOM mineralization if N and P are sufficient. Our study further demonstrates that structurally similar substrates can have distinct effects on SOM mineralization and the extent of SOM mineralization is strongly dependent on elemental stoichiometry, as well as the resource requirements of microbial decomposers.