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Nitrogen (N) additions often decrease soil respiration and increase soil organic carbon (C) stock. However, it is unclear how microbial substrates may shift with N additions and increasing temperature. Leveraging 12 years of N fertilization experiments and the associated shift in the dominant vegetation from C₄to C₃, we explored the δ¹³C‐CO₂and temperature sensitivities of respired CO₂and extracellular enzyme activities in control and fertilized soils. N additions increased cellulose‐decaying extracellular enzyme activity while respiration remained similar between the control and fertilized soils. Temperature sensitivity of cellulose‐decaying extracellular enzyme activity decreased with the N additions. The δ¹³C‐CO₂data reveal that, as temperature increased, microbes in fertilized soils changed their dominant substrate from bulk soil organic C to plant litterfall. Our results suggest that long‐term N fertilization imposed C limitation on microbes, leading to enhanced microbial efforts to acquire C. This study highlights how long‐term N additions can promote the relative preservation of organic C in mineral soil while litterfall, the precursor to mineral‐associated C, is increasingly decayed as temperatures increase.

Accurate representation of temperature sensitivity (Q₁₀) of soil microbial activity across time is critical for projecting soil CO₂efflux. As microorganisms mediate soil carbon (C) loss via exo‐enzyme activity and respiration, we explore temperature sensitivities of microbial exo‐enzyme activity and respiratory CO₂loss across time and assess mechanisms associated with these potential changes in microbial temperature responses. We collected soils along a latitudinal boreal forest transect with different temperature regimes (long‐term timescale) and exposed these soils to laboratory temperature manipulations at 5, 15, and 25°C for 84 days (short‐term timescale). We quantified temperature sensitivity of microbial activity per g soil and per g microbial biomass at days 9, 34, 55, and 84, and determined bacterial and fungal community structure before the incubation and at days 9 and 84. All biomass‐specific rates exhibited temperature sensitivities resistant to change across short‐ and long‐term timescales (meanQ₁₀ = 2.77 ± 0.25, 2.63 ± 0.26, 1.78 ± 0.26, 2.27 ± 0.25, 3.28 ± 0.44, 2.89 ± 0.55 for β‐glucosidase,N‐acetyl‐β‐d‐glucosaminidase, leucine amino peptidase, acid phosphatase, cellobiohydrolase, and CO₂efflux, respectively). In contrast, temperature sensitivity of soil mass‐specific rates exhibited either resilience (theQ₁₀value changed and returned to the original value over time) or resistance to change. Regardless of the microbial flux responses, bacterial and fungal community structure was susceptible to change with temperature, significantly differing with short‐ and long‐term exposure to different temperature regimes. Our results highlight that temperature responses of microbial resource allocation to exo‐enzyme production and associated respiratory CO₂loss per unit biomass can remain invariant across time, and thus, that vulnerability of soil organic C stocks to rising temperatures may persist in the long term. Furthermore, resistant temperature sensitivities of biomass‐specific rates in spite of different community structures imply decoupling of community constituents and the temperature responses of soil microbial activities.