Metabolic energy is required for a variety of functions in mammalian spermatozoa. However, this energy generation varies greatly between sperm of different species. Whilst the principle sources of energy are mitochondrial oxidative phosphorylation and glycolysis, each species has their own balance between these two pathways. The aim of this study was to compare relatively the aerobic metabolism between two species. The metabolism of each species was tested by forcing oxidative phosphorylation with a glucose free medium and it was important to determine if oxidative phosphorylation would lead to mitochondrial electron leakage; as this is known to cause oxidative stress and functional defects in sperm. Illustrated in the study is the significant role glycolysis plays in human spermatozoa, not only for supporting motility but also for their longevity. A lack of glucose or suitable substrate results in decreased motility and ATP production as well as an increase in mitochondrial ROS production. Suggesting that oxidative phosphorylation in human spermatozoa cannot generate sufficient levels of ATP to maintain motility and more notably can lead to electron leakage in the mitochondria. This generation of mitochondrial ROS has damaging downstream effects, including, 4-hydroxynonenal production, caspase activation and DNA damage. Whilst in equine spermatozoa, the opposite holds true. A lack of glucose or suitable substrate has no effects on motility, ATP production or ROS generation; indicating that oxidative phosphorylation in equine sperm is their main energy source. It also suggests that human mitochondria aren’t as well organized and capable for oxidative phosphorylation as equine spermatozoa. This study highlights how different mechanisms between species can play such different roles.