Cryopreservation of spermatozoa is a critical aspect of assisted reproduction. However in most cases, this process causes diminished spermatozoal function and low cellular survival. The effects of cryopreservation are further compounded in spermatozoa from individuals with disorders known to affect fertility. Teratospermia, characterized by the ejaculation of >60% morphologically abnormal spermatozoa, is one such disorder. Sperm from individuals with this condition exhibit reduced motility, impaired mitochondrial function, and a reduced ability to bind, penetrate, and fertilize an oocyte compared to sperm from normospermic males. Interestingly, even spermatozoa from teratospermic males that appear normal exhibit reduced functional capacity and following cryopreservation all cells are further impaired. More than 90% of felid species are affected by this condition, and it is prevalent among humans. Previous research suggested that impaired sperm metabolism in cheetahs – a ubiquitously teratospermic species – contributes to their poor cryosurvival and function, but the specific mechanisms are unknown. In this research, we hypothesized that inherent differences exist between the substrates and metabolic pathways utilized by sperm from normospermic and teratospermic individuals and on a species-specific basis. Gas chromatography - mass spectrometry (GC-MS) was used to investigate felid sperm metabolism using semen collected from domestic cats (Felis catus), cheetahs (Acinonyx jubatus), and clouded leopards (Neofelis nebulosa). The main objectives of this research were to: 1) characterize the metabolome of spermatozoa and seminal fluid; 2) investigate the role of β-oxidation in sperm motility and metabolism; and 3) interrogate the significance of specific metabolic pathways using metabolic activity profiling and heavy isotope tracers. Using GC-MS enabled the identification of metabolites which were unique to each species and sperm status (normospermic vs. teratospermic). Additionally, treatment of sperm with a β-oxidation inhibitor caused impaired motility in cheetah - but not domestic cat or clouded leopard - spermatozoa. Finally, fluxomic analysis demonstrated that glucose, fructose, and pyruvate are metabolized by felid sperm, but pathway utilization is species-specific. This is the first study to utilize a metabolomic and fluxomic approach to studying felid sperm, and the results of these studies illustrate the complexity of sperm metabolism on a species-specific basis. Understanding metabolic pathway activity in these cells will aid in the development of improved assisted reproduction techniques that may better facilitate sperm function and survival.