Aboveground plant litter and roots are primary sources of soil organic carbon (SOC), profoundly affecting soil respiration and SOC dynamics. With global change, the quantities of aboveground plant litter and root inputs into soil are changing in the subalpine region; however, the impacts of these changes on soil respiration and the quantity and quality of SOC remain unclear. Here, a three-year detritus input and removal treatment (DIRT) experiment that included six treatments (namely, control, CK; double litter, DL; litter removal, NL; root exclusion, NR; root exclusion plus double litter, NRDL; and root exclusion plus litter removal, NRNL) was conducted in a subalpine Cambisol on the eastern edge of the Tibetan Plateau to assess the different effects of aboveground litter and roots on soil respiration and the quantity and quality of SOC dynamics. Among the treatments, the mean soil respiration in NL, NR and NRNL treatments significantly decreased by 21.7%, 31.9% and 40.5%, respectively, compared to the CK treatment. When with intact roots, double litter (DL) had a positive impact on soil respiration, resulting in a 13.2% increase in soil respiration compared to the CK treatment. In contrast, when with root exclusion (in the absence of roots), combined root exclusion and litter addition (NRDL) hardly influence soil respiration compared with the NR treatment, resulting in a 30.5% decrease in soil respiration. The soil respiration, labile SOC content and the abundance of soil microbes exhibited similar patterns among treatments. Roots excluded (NR, NRDL, NRNL) exerted a stronger effects on soil respiration, temperature sensitivity of soil respiration (Q10), labile SOC content and the recalcitrance index of carbon (RIC) than aboveground plant litter manipulation (CK, DL, NL). Correlation analysis and redundancy analysis revealed that soil respiration and its Q10 were regulated by the quantity and quality of SOC substrates and the abundance of soil microbes. Our results highlighted that roots exerted greater influence on the quantity and quality of SOC, the abundance of soil microbes, soil respiration and its Q10 than aboveground litter manipulation in subalpine Cambisol in response to future climate scenarios, especially the mediating effect of the presence of roots on the function of aboveground plant litter.