<p>Soils are the only biological sink for atmospheric Methane (CH₄), making microbial methane consumption a dynamic component of the global carbon cycle. However, land-use change toward agriculture can inhibit this process, partly due to the application of NH₄⁺-based fertilizers. To assess whether this effect contributes to reductions in CH₄ uptake following land-use change in Brazilian biomes, we incubated soils from natural and cultivated areas across four endangered biomes: Caatinga, Cerrado, Pampas, and Atlantic Forest. Among soils exhibiting net CH₄ uptake, natural soils oxidized an average of 11.9&#xa0;µg C kg⁻¹ soil day⁻¹. Ammonium sulfate addition reduced CH₄ oxidation by 33% in pristine soils, whereas soils from agricultural fields showed no detectable methane uptake. With the exception of the Caatinga biome, pristine soils consumed atmospheric CH₄ and harbored higher abundances of methanotrophic bacteria compared to managed soils. Our findings indicate that nitrogen addition, at levels typical of NH₄⁺-based fertilization, had a less pronounced effect on CH₄ uptake than land-use change itself, underscoring the importance of conserving natural ecosystems to support the CH₄ cycle. Furthermore, soil nutrient availability, particularly micronutrients, emerged as a potential regulator of methanotrophy, capable of either stimulating or inhibiting microbial methane consumption. In the context of accelerated climate change in the tropics and globally, it is imperative to rethink agricultural practices, biomass production models, and land-use and fertilization strategies to minimize potential increases in greenhouse gas emissions.</p>