Understanding of effects of soil temperature and soil moisture on soil respiration (Rs) under future warming is critical to reduce uncertainty in predictions of feedbacks to atmospheric CO2 concentrations from grassland soil carbon. Intact cores with roots taken from a full factorial, 5-year alpine meadow warming and grazing experiment in the field were incubated at three different temperatures (i.e. 5, 15 and 25°C) with two soil moistures (i.e. 30 and 60% water holding capacity (WHC)) in our study. Another experiment of glucose-induced respiration (GIR) with 4 h of incubation was conducted to determine substrate limitation. Our results showed that high temperature increased Rs and low soil moisture limited the response of Rs to temperature only at high incubation temperature (i.e. 25°C). Temperature sensitivity (Q10) did not significantly decrease over the incubation period, suggesting that substrate depletion did not limit Rs. Meanwhile, the carbon availability index (CAI) was higher at 5°C compared with 15 and 25°C incubation, but GIR increased with increasing temperature. Therefore, our findings suggest that warming-induced decrease in Rs in the field over time may result from a decrease in soil moisture rather than from soil substrate depletion, because warming increased root biomass in the alpine meadow.