Temperature sensitivity (Q10) of soil organic matter (SOM) decomposition is an important parameter in models of the global carbon (C) cycle. Previous studies have suggested that substrate quality controls the intrinsic Q10, whereas environmental factors can impose large constraints. For example, physical protection of SOM and its association with minerals attenuate the apparent Q10 through reducing substrate availability and accessibility ([S]). The magnitude of this dampening effect, however, has never been quantified. We simulated theoretical Q10 changes across a wide range of [S] and found that the relationship between Q10 and the log10-transformed [S] followed a logistic rather than a linear function. Based on the unique Holocene paleosol chronosequence (7 soils from ca. 500 to 6900 years old), we demonstrated that the Q10 decreased nonlinearly with soil age up to 1150 years, beyond which Q10 remained stable. Hierarchical partitioning analysis indicated that an integrated C availability index, derived from principal component analysis of DOC content and parameters reflecting physical protection and mineral association, was the main explanatory variable for the nonlinear decrease of Q10 with soil age. Microbial inoculation and 13C-labelled glucose addition showed that low C availability induced by physical protection and minerals association attenuated Q10 along the chronosequence. A separate soil incubation experiment indicated that Q10 increased exponentially with activation energy (Ea) in the modern soil, suggesting that SOM chemical complexity regulates Q10 only when C availability is high. In conclusion, organic matter availability strongly decreased with soil age, whereas Michelis–Menten kinetics defines the Q10 response depending on C availability, but Arrhenius equation describes the effects of increasing substrate complexity. © 2022 John Wiley & Sons Ltd.