The microbiome function responses to land use change are important for the long-term prediction and management of soil ecological functions under human influence. However, it has remains uncertain how the biogeographic patterns of soil functional composition change when transitioning from natural steppe soils (NS) to agricultural soils (AS). We collected soil samples from adjacent pairs of AS and NS across 900 km of Mollisol areas in northeast China, and the soil functional composition was characterized using shotgun sequencing. AS had higher functional alpha-diversity indices with respect to KO trait richness and a higher Shannon index than NS. The distance-decay slopes of functional gene composition were steeper in AS than in NS along both spatial and environmental gradients. Land-use conversion from steppe to farmland diversified functional gene profiles both locally and spatially; it increased the abundances of functional genes related to labile carbon, but decreased those related to recalcitrant substrate mobilization (e.g., lignin), P cycling, and S cycling. The composition of gene functional traits was strongly driven by stochastic processes, while the degree of stochasticity was higher in NS than in AS, as revealed by the neutral community model and normalized stochasticity ratio analysis. Alpha-diversity of core functional genes was strongly related to multi-nutrient cycling in AS, suggesting a key relationship to soil fertility. The results of this study challenge the paradigm that the conversion of natural to agricultural habitat will homogenize soil properties and biology while reducing local and regional gene functional diversity.