ABSTRACT The effects of tillage on soil structure, physiology and microbiota structure were studied in a long-term field experiment, with side-to-side plots, established to compare effects of conventional tillage (CT) vs no-till (NT) agriculture. After 27 years, part of the field under CT was switched to NT and vice versa. Soil texture, soil enzymatic profiles and the prokaryotic community structure (16S rRNA genes amplicon sequencing) were analyzed at two soil depths (0–5 and 5–10 cm) in samples taken 6, 18 and 30 months after switching tillage practices. Soil enzymatic activities were higher in NT than CT, and enzymatic profiles responded to the changes much earlier than the overall prokaryotic community structure. Beta diversity measurements of the prokaryotic community indicated that the levels of stratification observed in long-term NT soils were already recovered in the new NT soils 30 months after switching from CT to NT. Bacteria and Archaea OTUs that responded to NT were associated with coarse soil fraction, soil organic carbon and C cycle enzymes, while CT responders were related to fine soil fractions and S cycle enzymes. This study showed the potential of managing the soil prokaryotic community and soil health through changes in agricultural management practices.