Temperature acclimation of soil organic matter (SOM) decomposition is one of the major uncertainties in predicting soil CO₂ efflux associated with the increase in global mean temperature. A reasonable explanation for an apparent acclimation proposed by Davidson and colleagues (2006) based on Michaelis-Menten kinetics suggests that temperature sensitivity decreases when both maximal activity of respiratory enzymes (V_max) and half-saturation constant (K_s) cancel each other upon temperature increase. We tested the hypothesis of the canceling effect by the mathematical simulation of data obtained in incubation experiments with forest and arable soils. Our data support the hypothesis and suggest that concentration of readily decomposable C substrate (as glucose equivalents) and temperature dependent substrate release are the important factors controlling temperature sensitivity of soil respiration. The highest temperature sensitivity of soil respiration was observed when substrate release was temperature dependent and C substrate concentration was much lower than K_s. Increase of substrate content to the half-saturation constant by glucose addition resulted in temperature acclimation associated with the canceling effect. Addition of the substrate to the level providing respiration at a maximal rate V_max leads to the acclimation of the whole microbial community as such. However, growing microbial biomass was more sensitive to the temperature alterations. This study improves our understanding of the instability of temperature sensitivity of soil respiration under field conditions, attributing this phenomenon to changes in concentration of readily decomposable C substrate.