Global change greatly alters the quality and quantity of plant litter inputs to soils and further impacts soil respiration. However, it is not fully understood how soil respiration may change with future shifts in litter input. The Detritus Input and Removal Treatment (DIRT) experiment were conducted in a coniferous forest (Platycladus orientalis (Linn.) Franco) ecosystem of central China to investigate the impact of above- and belowground litter input on soil respiration and the carbon-isotopic signature of soil-respired CO2. Short-term litter input manipulation significantly affected soil respiration. Based on annual flux values, soil respiration decreased by 31.9%, 20.5% and 37.2% in treatments with no litter (NL), no roots (NR) and no roots and no litter (NRNL), respectively, compared to the control (CK) treatment. Conversely, the double litter (DL) treatment increased soil respiration by 9.1% compared to the CK treatment. The recalcitrance index of carbon (RIC) and the relative abundance of fungi increased under NL, NR and NRNL treatment compared to the CK treatment. The carbon-isotopic signature of soil-respired CO2 was enriched under NRNL treatment and was slightly depleted under DL treatment compared to the CK treatment. The soil respiration rate and its carbon-isotopic signature exhibited similar seasonal patterns among treatments with higher soil respiration rates and lower d13C values of soil-respired CO2 in the summer compared with other seasons. Basal soil respiration was positively related to labile C and microbial biomass and negatively related to RIC and the fungi-to-bacteria (F:B) ratio, whereas the d13C value of soil-respired CO2 was negatively correlated with soil temperature and water content. Our results suggest that short-term litter input manipulation can affect soil respiration by altering substrate availability and microbial community structure and can impact the carbon-isotopic signature of soil-respired CO2 possibly due to changes in the components of soil respiration and soil microclimate.