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Extensive research has focused on the temperature sensitivity of microbial respiration in tundra and alpine meadows. However, the response of microbial respiration to thermal stress in alpine steppe soils, which have less organic matter and greater aeration, has received less attention. We investigated spatial and temporal variations in microbial respiration using an incubation experiment under different temperature (0, 15 and 30°C) and different percentages of water-holding capacity (WHC) (50 and 100%) conditions in the alpine steppe, and using a subsequent cooling experiment determined the ‘thermal stress' of soil microorganisms in response to increased temperature in the alpine steppe ecosystem. Microbial respiration rates decreased with increasing temperature at both 50 and 100% WHC for three sampling locations. Thermal stress of soil microorganisms under increased temperatures was found in the alpine steppe because subsequent cooling led to an increase in microbial respiration rates. Soil moisture did not affect microbial respiration, but the temperature sensitivity (Q10) of microbial respiration varied with the interactive effect between soil moisture and sampling location. Our findings suggest that the response of microbial respiration to high temperature may not be always positive as bacteria may experience thermal stress in the alpine steppe. Therefore, it is necessary to include thermal stress responses in alpine steppe models, as they may represent an important negative feedback effect on microbial respiration.