Understanding the temperature sensitivity (Q10) of soil organic matter (SOM) decomposition is important for predicting soil carbon (C) sequestration in terrestrial ecosystems under warming scenarios. Whether Q10 varies predictably with ecosystem succession and the ways in which the stoichiometry of input SOM influences Q10 remain largely unknown. We investigate these issues using a grassland succession series from free-grazing to 31-year grazing-exclusion grasslands in Inner Mongolia, and an incubation experiment performed at six temperatures (0, 5, 10, 15, 20, and 25°C) and with four substrates: control (CK), glucose (GLU), mixed grass leaf (GRA), and Medicago falcata leaf (MED). The results showed that basal soil respiration (20°C) and microbial biomass C (MBC) logarithmically decreased with grassland succession. Q10 decreased logarithmically from 1.43 in free-grazing grasslands to 1.22 in 31-year grazing-exclusion grasslands. Q10 increased significantly with the addition of substrates, and the Q10 levels increased with increase in N:C ratios of substrate. Moreover, accumulated C mineralization was controlled by the N:C ratio of newly input SOM and by incubation temperature. Changes in Q10 with grassland ecosystem succession are controlled by the stoichiometry of newly input SOM, MBC, and SOM quality, and the combined effects of which could partially explain the mechanisms underlying soil C sequestration in the long-term grazing-exclusion grasslands in Inner Mongolia, China. The findings highlight the effect of substrate stoichiometry on Q10 which requires further study.