Changes in soil respiration, one of the major fluxes of global carbon cycling, could significantly slow down or accelerate the increase in atmospheric CO2, with consequent feedbacks to climate change. It is critical to understand how substrate availability regulates soil respiration in projecting the response of carbon cycling to changed climate. We conducted a clipping and shading experiment for 1 year in a tallgrass prairie of the Great Plains, United States, to manipulate substrate supply to soil respiration. Our results showed that reduced substrate supply under clipping and/or shading significantly decreased soil respiration at all the timescales (diurnal, transient, and annual) irrespective of the minor concurrent changes in soil temperature and moisture. Annual mean soil respiration decreased significantly by 33, 23, and 43% for the clipping, shading, and clipping plus shading treatments, respectively. Temperature sensitivity of soil respiration decreased from 1.93 in the control plots to 1.88, 1.75, and 1.83 in the clipped, shaded, and clipped plus shaded plots, respectively. Rhizosphere respiration, respiration from decomposition of aboveground litter, and respiration from oxidation of soil organic matter and dead roots accounted for 30, 14, and 56% of annual mean soil respiration, respectively. Rhizosphere respiration was more sensitive to temperature than the other two components. Our results suggest a critical role of substrate supply in regulating soil respiration and its temperature sensitivity.