Diet-induced obesity (DIO) is a significant health concern which has been linked to structural and functional changes in the gut microbiota. Exercise (Ex) is effective in preventing obesity, but whether Ex alters the gut microbiota during development with high fat (HF) feeding is unknown. The objective of this study was to determine the effects of voluntary Ex on the gastrointestinal microbiota in LF-fed mice and in HF-DIO.