Livestock grazing is a global land-use activity with multiple social, cultural, and environmental impacts. However, it is expected to decrease progressively in the Cantabrian Mountain areas as notable changes in livestock management systems have been observed, such as shorter stay in mountain, lower stocking rates, and less shepherd control. This study aimed to evaluate the effect of grazing abandonment on microbial function and diversity through changes promoted in aboveground vegetation and soil properties. In 2005, we erected permanent fences around two 50 Â 50-m plots (excluded plots) in a temperate mountain grassland that has historically supported livestock managed by an extensive grazing system. A grazed plot was defined around each excluded plot. After 5 years, grazing abandonment induced shifts in floristic composition, decreased soil compaction at 0–10 cm soil depth, and reduced soil temperature in the summer due to a thicker plant layer. Consequently, microorganisms reduced soil enzymatic activity and microbial biomass, and increased CO 2 emissions and metabolic quotient, which indicated a lower metabolic efficiency of soil processes in excluded plots. These results suggest that soil microbial community function was very sensitive to the impacts of livestock grazing exclusion. The bacterial community was more diverse compared to the fungal community, but no significant difference in bacterial species richness was found between excluded and grazed plots. Microbial genetic diversity was not directly correlated with aboveground vegetation diversity and no clear pattern emerged as a response to grazing abandonment, probably because soil microbial diversity depends on site attributes that operate at a very fine spatial scale.