Accelerating global warming causes positive soil carbon (C) feedbacks to the atmosphere, yet the underlying microbial and physical mechanisms are still unclear. Microbial C use efficiency (CUE) and physical protection of soil organic matter (SOM) via organo-mineral interactions have been proposed to regulate microbial feedbacks to the climate system. We hypothesize that warming-altered physical protection may affect microbial activity and CUE, and fundamentally influence soil-climate feedbacks. Thus, soils were collected at the Prospect Hill of Harvard Forest, where soils have been warmed 5 °C above ambient for 27 years. Macroaggregates (250 – 2000 µm) and microaggregates (< 250 µm) were separately incubated with substrates (glucose, cellobiose and cellulose), or as intact or crushed aggregates at 15 °C or 25 °C for 24 h. Samples were 18O-water labeled (20 atm%) to calculate CUE. Microbial biomass C (MBC), soil organic C (SOC), total nitrogen (N), dissolved organic C (DOC), respiration, and soil DNA were analyzed. Our results show that: 1) Long-term warming decreased soil nutrient content, substrate availability, microbial biomass and activity based on reduced soil N, MBC, SOC, DOC, respiration, and DNA content. Warming also increased metabolic quotient, namely microbes respired more C per unit of MBC, indicating a lower microbial CUE. This is consistent with observed trends in increased CO2 loss due to long-term warming. 2) Microaggregates have increased soil nutrient content and C storage, with greater soil N, MBC, DOC, DNA content, and decreased metabolic quotient, suggesting a higher CUE. 3) Higher incubation temperature increased respiration, DNA content, and metabolic quotient, indicating a lower CUE. 4) Substrate quality did not affect soil C loss or microbial activity. Adding substrates showed little effect on respiration and metabolic quotient in the 24 h incubation. 5) Crushed aggregates reduced soil MBC but increased DOC, respiration, and metabolic quotient, suggesting that physically unprotected SOM has a lower CUE and is made more vulnerable to microbial degradation over long-term warming. To conclude, our findings show that aggregate size and integrity are important regulators of the size of microbial biomass and microbially available C, indicating that future experiments should focus on defining the microbial actors and mechanisms, which could be crucial in predicting soil C feedbacks in a warmer world.