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Adaptive multi-paddock (AMP) grazing is a form of rotational grazing in which small paddocks are grazed with high densities of livestock for short periods, with long recovery periods prior to regrazing. We compared the fluxes of greenhouse gases (GHGs), including carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O), from soils of AMP-grazed grasslands to paired neighboring non-AMP-grazed grasslands across a climatic gradient in Alberta, Canada. We further tested GHG responses to changes in temperature (5 °C vs. 25 °C) and moisture levels (permanent wilting point (PWP), 40% of field capacity (0.4FC), or field capacity (FC)) in a 102-day laboratory incubation experiment. Extracellular enzyme activities (EEA), microbial biomass C (MBC) and N (MBN), and available-N were also measured on days 1, 13, and 102 of the incubation to evaluate biological associations with GHGs. The 102-day cumulative fluxes of CO2, N2O, and CH4 were affected by both temperature and moisture content (p < 0.001). While cumulative fluxes of N2O were independent of the grazing system, CH4 uptake was 1.5 times greater in soils from AMP-grazed than non-AMP-grazed grasslands (p < 0.001). There was an interaction of the grazing system by temperature (p < 0.05) on CO2 flux, with AMP soils emitting 17% more CO2 than non-AMP soils at 5 °C, but 18% less at 25 °C. The temperature sensitivity (Q10) of CO2 fluxes increased with soil moisture level (i.e., PWP < 0.4FC ≤ FC). Structural equation modelling indicated that the grazing system had no direct effect on CO2 or N2O fluxes, but had an effect on CH4 fluxes on days 1 and 13, indicating that CH4 uptake increased in association with AMP grazing. Increasing soil moisture level increased fluxes of GHGs—directly and indirectly—by influencing EEAs. Irrespective of the grazing system, the MBC was an indirect driver of CO2 emissions and CH4 uptake through its effects on soil EEAs. The relationships of N-acetyl-β glucosaminidase and β-glucosidase to N2O fluxes were subtle on day 1, and independent thereafter. AMP grazing indirectly affected N2O fluxes by influencing N-acetyl-β glucosaminidase on day 13. We conclude that AMP grazing has the potential to mitigate the impact of a warmer soil on GHG emissions by consuming more CH4 compared to non-AMP grazing in northern temperate grasslands, presumably by altering biogeochemical properties and processes. [ABSTRACT FROM AUTHOR]