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Grasslands are used extensively for grazing livestock, and variation in grazing management may affect the soil microbial community and ecosystem functions, such as nutrient cycling, in grasslands. In particular, adaptive multi-paddock (hereafter 'multi-paddock') grazing is considered a regenerative grazing management practice that can improve the sustainability of grasslands. However, little is known about how multi-paddock grazing affects the soil microbial community, which plays an importance role in global biogeochemical cycling. In this study, grassland soils were collected from 15 ranch pairs located across the Canadian prairie, where, in each pair, one ranch practiced multi-paddock grazing while the other practiced conventional grazing (varying from continuous to slow to fast rotational grazing). We used soil phospholipid fatty acid (PLFA) profiles to quantify microbial functional groups, and measured microbial biomass C and soil properties. Our data show that microbial PLFA abundances and microbial biomass C was not different between the two grazing systems. However, multi-paddock grazing resulted in lower ratios of microbial biomass C: N (MBC:MBN), fungi: bacteria (F:B) and Gram-positive: Gram-negative bacteria in grassland soils compared to conventional grazing. Further investigation of specific management metrics revealed that MBC:MBN and F:B ratios were most affected by cattle stocking rate and stocking density, respectively. Thus, there is potential to affect soil function by altering the composition of soil microbial communities through multi-paddock grazing. • Adaptive multi-paddock grazing (AMP) altered soil microbial PLFA composition. • Increased cattle stocking rates reduced microbial biomass C:N. • Increased cattle stocking density decreased fungi:bacteria ratio. • AMP reduced MBC:MBN, F:B and Gram-positive:Gram-negative ratios. • Microbial changes indicate potential effects on carbon cycling and storage. [ABSTRACT FROM AUTHOR]