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Improved mechanistic understanding of soil methane (CH₄) exchange responses to shifts in soil moisture and temperature in forest ecosystems is pivotal to reducing uncertainty in estimates of the soil-atmospheric CH₄ budget under climate change. We investigated the mechanism behind the effects of soil moisture and temperature shifts on soil CH₄ fluxes under laboratory conditions. Soils from the Huai River Basin in China, an area that experiences frequent hydrological shifts, were sampled from two consecutive depths (0–20 and 20–50 cm) and incubated for 2 weeks under different combinations of soil moisture and temperature. Soils from both depths showed an increase in soil moisture and temperature-dependent cumulative CH₄ fluxes. CH₄ production rates incubated in different moisture and temperature in surface soil ranged from 1.27 to 2.18 ng g⁻¹ d⁻¹, and that of subsurface soil ranged from 1.18 to 2.34 ng g⁻¹ d⁻¹. The <i>Q</i>₁₀ range for soil CH₄ efflux rates was 1.04–1.37. For surface soils, the relative abundance and diversity of methanotrophs decreased with moisture increase when incubated at 5 °C, while it increased with moisture increase when incubated at 15 and 30 °C. For subsurface soils, the relative abundance and diversity of methanotrophs in all samples decreased with moisture increase. However, there was no significant difference in the diversity of methanogens between the two soil depths, while the relative abundance of methanogens in both depths soils increased with temperature increase when incubated at 150% water-filled pore space (WFPS). Microbial community composition exhibited large variations in post incubation samples except for one treatment based on the surface soils incubated at 15 °C, which showed a decrease in the total and unique species number of methanotrophs with moisture increase. In contrast, the unique species number of methanogens in surface soils increased with moisture increase. The analysis of distance-based redundancy analysis (db-RDA) showed that soil pH, dissolved organic carbon (DOC), dissolved organic nitrogen (DON), microbial biomass carbon (MBC), NO₃⁻-N, and NH₄⁺-N mainly performed a significant effect on methanotrophs community composition when incubated at 60% WFPS, while they performed a significant effect on methanogens community composition when incubated at 150% WFPS. Overall, our findings emphasized the vital function of soil hydrology in triggering CH₄ efflux from subtropical plantation forest soils under future climate change.