Adenosine triphosphate (ATP) production via glycolysis and oxidative phosphorylation is essential for the maintenance of flagellar motility in sperm; however, the primary energy production pathways supporting fertilization vary among species. Inconsistency in thought exists regarding which pathways maintain ATP production and sperm motility in poultry. Glycolysis and mitochondrial oxidation contribute to flagellar motion in chicken sperm, but the relative dependence on these pathways for motility and penetrability into the inner perivitelline layer remains unclear. In the present study, there was use of various inhibitors and energy substrates to evaluate the relative contribution of anaerobic glycolysis and mitochondrial oxidation to chicken sperm flagellar motility, ATP production, and penetrating capacity through the perivitelline layer. Although both pathways contributed to these processes to varying extent, glucose was the primary substrate for sperm penetration into the inner perivitelline layer in chickens. Furthermore, results from metabolic stress analyses indicated that there was less perivitelline penetrability in response to pyruvate that was not due to changes in reactive oxygen species or intracellular pH. Overall, results from the present study indicate glycolysis and mitochondrial oxidation pathways have distinct functions in the flagellar motility and penetrability of the perivitelline membrane by rooster sperm. There, therefore, are new insights as a result of findings in the present study into the energy production system of sperm through which there is utilization of extracellular metabolic substrates for maintaining sperm fertilization capacity.