Gut microbiota have been associated with health, disease and behaviour in several species and are an important link in gut-brain axis communication. Diet plays a key role in affecting the composition of gut microbiota. In horses, high-starch diets alter the hindgut microbiota. High-starch diets are also associated with increased behavioural reactivity in horses. These changes in microbiota and behaviour may be associated. This study compares the faecal microbiota and behaviour of 10 naïve ponies. A cross-over design was used with experimental groups fed high-starch (HS) or high-fibre (HF) diets. Results showed that ponies were more reactive and less settled when being fed the HS diet compared to the HF diet. Irrespective of diet, the bacterial profile was dominated by two main phyla, Firmicutes, closely followed by Bacteroidetes. However, at lower taxonomic levels multivariate analysis of 16S rRNA gene sequencing data showed diet affected faecal microbial community structure. The abundance of 85 OTUs differed significantly related to diet. Correlative relationships exist between dietary induced alterations to faecal microbiota and behaviour. Results demonstrate a clear link between diet, faecal microbial community composition and behaviour. Dietary induced alterations to gut microbiota play a role in affecting the behaviour of the host.