Regenerative agriculture (RA) is expanding across the Western Canadian Prairies, but its microbial foundations under climatic constraint remain insufficiently integrated. This review synthesizes evidence from long-term Prairie field experiments, regional and global datasets to evaluate how regenerative management reshapes soil biological processes and agronomic performance across systems. RA practices including no-till, diversified rotations, cover cropping, and organic amendments consistently enhance microbial biomass (up to 40–86%), arbuscular mycorrhizal fungal abundance (32–60%), and microbial diversity (≈50%), alongside increases in soil organic carbon (up to 15.6 kg C ha⁻¹ yr⁻¹), aggregate stability (up to 38%), and water retention (up to 30–34%). These biologically mediated improvements are linked to enhanced nutrient cycling and crop nitrogen uptake (13–47%), as well as increased microbial enzymatic activity and functional gene abundance. Agronomically, these changes translate into yield gains ranging from 10% to 147% under long-term no-till and 14–38% under diversified rotations, with additional system-level benefits including reductions in synthetic nitrogen inputs (up to 73%) and herbicide use (up to 42%). While agronomic benefits vary across temporal scales and environmental conditions, this synthesis identifies microbial communities as key mediators of interactions among climate, plant, and soil systems, underpinning improvements in soil health, pest suppression, and yield stability in semi-arid, climate-variable Prairie agroecosystems. Continued long-term, system-level research is needed to refine regionally adapted regenerative transitions and to clarify how microbial processes mediate resilience under future climate uncertainty.