Soil organic matter (SOM) dynamics ultimately govern the ability of soil to provide long-term C sequestration and the nutrients required for ecosystem productivity. Predicting belowground responses to elevated CO2 requires an integrated understanding of SOM transformations and the microbial activity that governs them. It remains unclear how the microorganisms upon which these transformations depend will function in an elevated CO2 world. This study examines SOM transformations and microbial metabolism in soils from the Duke Free Air Carbon Enrichment site in North Carolina, USA. We assessed microbial respiration and net nitrogen (N) mineralization in soils with and without elevated CO2 exposure during a 100-day incubation. We also traced the depleted C isotopic signature of the supplemental CO2 into SOM and the soils' phospholipid fatty acids (PLFA), which serve as biomarkers for living cells. Cumulative net N mineralization in elevated CO2 soils was 50% that in control soils after a 100-day incubation. Respiration was not altered with elevated CO2. C : N ratios of bulk SOM did not change with elevated CO2, but incubation data suggest that the C : N ratios of mineralized organic matter increased with elevated CO2. Values of SOM δ13C were depleted with elevated CO2 (−26.7±0.2 vs. −30.2±0.3‰), reflecting the depleted signature of the supplemental CO2. We compared δ13C of individual PLFA with the δ13C of SOM to discern incorporation of the depleted C isotopic signature into soil microbial groups in elevated CO2 plots. PLFA i15:0, a15:0, and 10Met18:0 reflected significant incorporation of recently produced photosynthate, suggesting that the bacterial groups defined by these biomarkers are active metabolizers in elevated CO2 soils. At least one of these groups (actinomycetes, 10Met18:0) specializes in metabolizing less labile substrates. Because control plots did not receive an equivalent 13C tracer, we cannot determine from these data whether this group of organisms was stimulated by elevated CO2 compared with these organisms in control soils. Stimulation of this group, if it occurred in the elevated CO2 plot, would be consistent with a decline in the availability of mineralizable organic matter with elevated CO2, which incubation data suggest may be the case in these soils.