It is crucial to promote soil carbon sequestration while reducing CO2 emissions to mitigate climate change. However, the extent of increasing actual soil carbon storage depends on the amount and composition of organic matter input, including its fate during decomposition and soil organic matter (SOM) formation via microbial transformation. With respect to the need to increase carbon sequestration in soil and sustain soil fertility, it is of great interest to better understand how soils with different organic matter content react to amendment with fresh organic matter. Here, we incubated three agricultural soils representing a gradient in C content, adding two different 13C labeled plant residues varying in carbon-to-nitrogen ratio. Carbon mineralization was monitored together with the analysis of the 13CO2 signatures. After the incubation, 13C compound-specific PLFAs, microbial necromass, and enzyme activities were analyzed. This study demonstrates that the carbon return on investment, thus the amount of retained fresh carbon in relation to the amount of added organic matter, clearly depends on the amount of native soil carbon. Notably, the addition of fresh organic matter to carbon-deficient soils leads to a higher specific CO2 release compared to soils with high carbon loading, which can be attributed to the differences in the soil microorganisms' response. The CO2 release of the soil with the lowest C-content was 2.1 and 2.0 mg g−1 soil for treatment with oat and pea litter addition, respectively, whereas for the soil with the highest C-content, CO2 release was 1.7 mg g−1 soil for oat treatment and 1.6 mg g−1 soil for pea treatment. Thus, higher SOC contents sustain a higher ‘return on investment’ for the fresh carbon that is amended to soils. With plant litter amendments the microbial community shifted towards a higher fungi-to-bacteria ratio (F/B). This shift in the microbial community was more pronounced (F/B ranging from 0.04 to 0.11) with the addition of oat litter (low quality) compared to pea litter (high quality). Hence, it is important to consider the fate of organic amendments with different N availability when aiming to rebuild soil carbon stocks in degraded soils. Soil management should focus on sustaining soil carbon in balance with current carbon stocks to avoid the vicious circle of soils losing carbon in conjunction with increased greenhouse gas release.