Abstract: To investigate the effects of cultivation duration of Ganoderma lucidum on soil organic carbon fractions and microbial functional characteristics, this study focused on G. lucidum forest plots with different cultivation durations（2, 4, and 6 years）, using native broadleaf forest as the control（CK）. Soil samples were collected from the 0–20 cm layer to determine the contents of different forms of soil organic carbon. Microbial metabolic activity and functional diversity were assessed using Biolog EcoPlate™ technology. The results showed that understory G. lucidum cultivation had no significant effect on soil total organic carbon（TOC）content. However, with increasing cultivation duration, soil microbial biomass carbon（MBC）and water-soluble organic carbon（WSOC）contents exhibited an initial increase followed by stabilization. Specifically, the MBC and WSOC contents in the 2-year treatment were significantly higher than those in the other treatments（P＜0.05）, increasing by 29.99% and 28.67% compared to CK, respectively. Microbial metabolic activity, represented by the average well color development（AWCD）value, peaked in the 2-year treatment, with AWCD values in CK and the 2-year treatment being significantly higher than those in the 4-year and 6-year treatments（P＜0.05）. Moreover, functional diversity indices, including the Shannon-Wiener diversity index, Simpson dominance index, McIntosh evenness index, and Pielou evenness index, were also highest in the 2-year treatment, indicating a significant enhancement of microbial functional diversity during the initial cultivation stage. Principal component analysis revealed distinct patterns of carbon source utilization by soil microbial communities across different cultivation durations, with carbohydrate carbon sources being the key factor distinguishing G. lucidum cultivated and uncultivated soils. Correlation analysis further indicated positive correlations between all microbial functional diversity indices and TOC, MBC, and WSOC. In conclusion, understory G. lucidum cultivation exhibits a soil ecological effect characterized by“short-term promotion and long-term inhibition.” It is recommended to implement intermittent cultivation or fallow systems to maintain soil health and ensure the sustainability of understory cultivation practices.