Horses can suffer from gastrointestinal (GI) disease in domestic environments, often precipitated by human‐led changes in management. Understanding the consequences of these changes on equine gut microbiota is key to the prevention of such disease episodes.

Profile the faecal microbiota of adult female Exmoor ponies under three management conditions, representing increasing levels of management by humans, encompassing different diets; whilst controlling for age, breed and sex.

Cross‐sectional descriptive.

Faecal samples were collected from three populations of Exmoor ponies kept under contrasting management conditions: 29 adult female ponies in groups with low management (LM) (n = 10), medium management (MM) (n = 10) and high management (HM) (n = 9) levels, based on diet, drug use, handling and exercise. Faecal microbial composition was profiled via high‐throughput sequencing of the bacterial 16S rRNA gene, and functional metagenome predictions.

We observed profound step‐wise changes in microbiome structure in the transition from LM to MM to HM. A relatively high abundance of Proteobacteria and Tenericutes was associated with the HM group; higher abundance of Methanobacteria was observed in the LM group. The MM group had intermediate levels of these taxa and exhibited high ‘within group’ variation in alpha diversity. Functional predictions revealed increased amino acid and lipid metabolism in HM; energy metabolism in LM and carbohydrate metabolism and immune/metabolic disease pathways in MM.

Low group sizes, incomplete knowledge of bacterial genomes in equine gut microbiota and it was not possible to assess the relative impact of diet, drug use, handling and exercise on the microbiome as variables were confounded.

Human‐led management factors had profound step‐wise effects on faecal microbial composition. Based on functional metagenome predictions, we hypothesise that dietary differences between groups were the major driver of observed differences.