<p>Substrate concentration is a key determinant of temperature sensitivity (<i>Q</i>₁₀) in soil organic matter (SOM) decomposition. Although the microbial processes underlying this relationship are well documented, the role of soil fauna, particularly earthworms, in shaping the temperature response of SOM decomposition remains largely unexplored. In this study, we examined how earthworms and their deposits (excreta and secretions) influence the SOM decomposition rate (<i>R</i>_s) and <i>Q</i>₁₀ across natural substrate concentration gradients. We combined stratified sampling across a 1-m soil profile (representing vertical variation in substrate concentration gradients) with laboratory incubations under simulated diurnal temperature fluctuations. Using a high-frequency (CO₂ concentration captured at 1-s intervals) automated system, we quantified the effects of earthworms and their deposits on <i>R</i>_s and <i>Q</i>₁₀. The results showed that earthworm deposits alone moderately increased <i>R</i>_s by 22–56% and <i>Q</i>₁₀ by 4–14%, with stronger effects in surface soils characterized by high substrate concentrations. Earthworm continued presence substantially amplified these responses, increasing <i>R</i>_s by 194–277% and <i>Q</i>₁₀ by 26–63%, notably, the greatest increase in <i>Q</i>₁₀ occurred under low substrate concentrations. Earthworm activity strengthened the dependence of <i>Q</i>₁₀ on both substrate concentration and microbial activity; however, earthworm endogenous <i>Q</i>₁₀ remained stable across substrate and temperature conditions. These findings demonstrate that earthworms are strong biotic regulators of the temperature sensitivity of SOM decomposition, particularly in substrate-limited soils.</p>