The grassland biome is an important sink for CH₄, a major greenhouse gas. There is considerable uncertainty in the grassland CH₄ sink capacity due to diverse environmental gradients in which grasslands occur, and many environmental conditions can affect abiotic (e.g., CH₄ diffusivity into soils) and biotic (e.g., methanotrophy) factors that determine spatial and temporal CH₄ dynamics. We investigated the relative importance of a soil’s gas diffusivity vs. net methanotroph activity in 22 field plots in seven sites distributed across the US Great Plains by making ca. bi-weekly measures during the growing seasons over three years. We quantified net methanotroph activity and diffusivity by using an approach combining a gas tracer, chamber headspace measurements, and a mathematical model. At each plot, we also measured environmental characteristics, including water-filled pore space (WFPS), soil temperature and inorganic nitrogen contents, and examined the relative importance of these for controlling diffusivity and net methanotroph activity. At most of the plots across the seven sites, CH₄ uptake rates were consistently greatest when WFPS was intermediate at the plot level. Our results show that variation in net methanotroph activity was more important than diffusivity in explaining temporal variations in net CH₄ uptake, but the two factors were equally important for driving spatial variation across the seven sites.  Water-filled pore space was a significant predictor for diffusivity only in plots with sandy soils. Water-filled pore space was the most important control on net methanotroph activity, with net methanotroph activity showing a parabolic response to WFPS (concave down), and the shape of this response differed significantly among sites. Moreover, we found that the WFPS level at peak net methanotroph activity was strongly correlated with mean annual precipitation of the site. These results suggest that local precipitation regime determines unique sensitivity of CH₄ uptake rates to soil moisture. Our findings indicate that grassland CH₄ uptake may be predicted using local soil water conditions. More variable soil moisture, potentially induced through predicted future extremes of rainfall and drought, could reduce grassland CH₄ sink capacity in the future.