The gut microbiota plays an important role in host physiology. However, the effects of host sex, lifestyle, and temporal influences on the bacterial community within the gut remain ill-defined. To address this gap, we evaluated 56 male and female mice over a 10-week study to assess the effects of sex, diet, and exercise on gut community dynamics. Mice were randomly assigned to high-fat or control diet feeding and had free access to running wheels or remained sedentary throughout the study period. The fecal bacterial community was characterized by rRNA operon amplicon profiling via nanopore sequencing. Differential abundance testing indicated that ~200 bacterial taxa were significantly influenced by sex, diet, or exercise (4.2% of total community), which also changed over time (82 taxa, 1.7% of total community). Phylogenetic analysis of taxa closely related to Dysosmobacter welbionis and several members of the family Muribaculaceae were examined more closely and demonstrated distinct species/strain-level sub-clustering by host sex, diet, and exercise. Collectively, this data suggests that sex and lifestyle can alter the gut bacteriota at the species/strain-level which may play a role in host health. These results also highlight the need for improved characterization methods to survey microbial communities at finer taxonomic resolution.