Soil respiration is an important part of the global carbon (C) cycle and the largest component of C flux from terrestrial ecosystems to the atmosphere. Here, we investigated possible effects of photosynthetic substrate supply on soil respiration in a semiarid ecosystem. A field experiment combining water addition and shading (low and high shading) treatments was conducted to manipulate photosynthetic substrate supply in a temperate semiarid steppe in two growing seasons. Our result showed that water addition and/or low shading significantly increased net primary productivity (ecosystem-level photosynthetic substrate supply) and soil respiration in both two growing seasons. However, the effects of high shading on net primary productivity and soil respiration depended on soil water condition, which were negative in wet year (2008) but positive in dry year (2009). On the diel timescale, soil respiration was out of phase with soil temperature and leaf net photosynthesis, but in phase with leaf sugar and starch contents (leaf-level photosynthetic substrate production). The results indicated that photosynthetic substrate supply was an important factor in regulating soil respiration on both daily and seasonal timescales. Moreover, its effect on soil respiration increased with increasing water availability in this region. The predominant role of C assimilate supply on soil respiration indicates that the predicted positive influence of rising temperature on soil respiration will be simultaneously mediated by substrate supply and water availability in semiarid steppe ecosystems.