Sphagnum cultivation has several ecological environmental effects, including land restoration, and economic benefits. However, complex mechanisms involving the interaction of microbial structure, functions, and nutrient limitations under Sphagnum cultivation are still unclear. In this study, we explored microbial nutrient limitation by examining the soil extracellular enzyme stoichiometry across different years of Sphagnum cultivation and further analyzed the impacts of Sphagnum cultivation on soil properties, microbial communities, and microbial functional genes. The results revealed that microbial carbon (C) limitation increased with the years of Sphagnum cultivation. However, Sphagnum cultivation alleviated microbial phosphorus (P) limitation. Sphagnum cultivation significantly decreased the microbial diversity, and microorganisms shifted from copiotrophic (r-) to oligotrophic (K-) groups, which was consistent with the observed reduction in the 16S rRNA operon copy number within the microbial community. However, the functions related to C, nitrogen (N), P, and sulfur cycles displayed a similar “decrease–peak–increase” trend across different years of Sphagnum cultivation. Microbial C limitation increased mainly through increasing the expression of functional genes involved in C cycling, whereas microbial P limitation decreased mainly through decreasing the expression of functional genes involved in N and P cycling with the years of Sphagnum cultivation. Therefore, microbial functional attributes were the key factors driving the response of soil microbial metabolic constraints to the chronosequences of Sphagnum cultivation. Additionally, soil organic C (SOC) content increased and total N and total P contents first decreased and then increased with the years of Sphagnum cultivation. The SOC content was significantly higher by 110.9 % and 178.8 % on average in paddies under long-term Sphagnum cultivation (10 and 20 years, respectively) than in those without Sphagnum cultivation. These findings demonstrated the positive influence of Sphagnum farming on soil ecosystems, particularly in boosting organic C sequestration, which is crucial for combating global climate change.