Soil pH significantly impacts microbial activity and community assembly, which in turn determines the temperature sensitivity (Q₁₀) of soil respiration. Due to the high soil acidification in China, it is necessary to understand how soil acidification impacts Q₁₀. Here, the Q₁₀ of soil respiration was examined in a long-term field experiment (1982–present) with different soil pH caused by fertilization management. In this experiment, we selected treatments with neutral pH: (1) no crops and fertilization (CK); (2) crops without fertilization (NF); low pH with (3) crops with chemical fertilization (NPK); and (4) crops with chemical fertilization combined with wheat straw incorporation (WS). Under natural soil temperature changes, we observed that soil acidification lowered the Q₁₀ value of soil respiration. Considering only temperature changes, the Q₁₀ of soil respiration was strongly associated with microbial community composition, alpha diversity, and soil ammonium nitrogen. Considering the interaction between soil pH and temperature, warming strengthened the negative effect of soil pH on the Q₁₀ of soil respiration, and the pathway through which soil pH mediated Q₁₀ included not only microbial community composition, alpha diversity, and biomass but also the soil’s available phosphorus. This work enhanced our insights into the relationships between Q₁₀, temperature, and soil pH by identifying important microbial properties and key soil environmental factors.