Mitochondrial dysfunction is prevalent in the ageing gastrointestinal tract. We investigated whether mitochondrial function in ageing colonic crypts and exercise influence microbial gut communities in mice. Twelve PolgAmut/mut mice were randomly divided into a sedentary and exercise group at 4 months. Seven aged matched PolgA+/+ mice remained sedentary throughout. Stool samples were collected at 4, 7 and 11 months, and bacterial profiling was achieved through 16S rRNA sequencing profiling. Mitochondrial enzyme activity was assessed in colonic epithelial crypts at 11 months for PolgAmut/mut and PolgA+/+ mice. Sedentary and exercised PolgAmut/mut mice had significantly higher levels of mitochondrial dysfunction than PolgA+/+ mice (78%, 77% and 1% of crypts, respectively). Bacterial profiles of sedentary PolgAmut/mut mice were significantly different from the sedentary PolgA+/+ mice, with increases in Lactobacillus and Mycoplasma, and decreases in Alistipes, Odoribacter, Anaeroplasma, Rikenella, Parabacteroides, Allobaculum in the PolgAmut/mut mice. Exercise did not have any impact upon gut mitochondrial dysfunction, however, exercise did increase gut microbiota diversity and significantly increasing bacterial genera Mucispirillum and Desulfovibrio. Mitochondrial dysfunction is associated with changes in the gut microbiota. Endurance exercise moderated some of these changes, establishing that environmental factors can influence gut microbiota despite mitochondrial dysfunction.