Simple SummaryGlobal mean temperature has increased by 1.07 °C since the Industrial Revolution, which arouses people’s widespread concern about climate warming. Nighttime temperatures increase faster and higher than daytime temperatures around the world. Various microbial groups in the soil may respond differently to such asymmetrically diurnal warming and then change ecological functions. In this study, we used a ten-year experiment in a semi-arid grassland to examine the effects of daytime and nighttime warming on soil microbial composition. The results showed that short-term warming did not change soil microbial composition, but long-term daytime warming rather than nighttime warming decreased the fungi-to-bacteria ratio in soils. In addition, soil respiration enhanced with the decreasing fungi-to-bacteria ratio. This work implies the importance of soil microbial composition in regulating grassland C release under long-term warming, which may help us accurately assess the climate-C feedback in terrestrial ecosystems.AbstractClimate warming has profoundly influenced community structure and ecosystem functions in the terrestrial biosphere. However, how asymmetric rising temperatures between daytime and nighttime affect soil microbial communities that predominantly regulate soil carbon (C) release remains unclear. As part of a decade-long warming manipulation experiment in a semi-arid grassland, we aimed to examine the effects of short- and long-term asymmetrically diurnal warming on soil microbial composition. Neither daytime nor nighttime warming affected soil microbial composition in the short term, whereas long-term daytime warming instead of nighttime warming decreased fungal abundance by 6.28% (p< 0.05) and the ratio of fungi to bacteria by 6.76% (p< 0.01), which could be caused by the elevated soil temperature, reduced soil moisture, and increased grass cover. In addition, soil respiration enhanced with the decreasing fungi-to-bacteria ratio, but was not correlated with microbial biomass C during the 10 years, indicating that microbial composition may be more important than biomass in modulating soil respiration. These observations highlight the crucial role of soil microbial composition in regulating grassland C release under long-term climate warming, which facilitates an accurate assessment of climate-C feedback in the terrestrial biosphere.