Mammalian sperm require a long period of time to accomplish fertilization, and thus they must produce energy (ATP) by metabolizing energy substrates. Mammalian sperm can produce ATP by two pathways, glycolysis and mitochondrial respiration. Among them, glycolysis is now widely considered to be a key pathway in mammalian sperm energy production. However, correlation between glycolysis and mammalian sperm motility has not yet been fully elucidated. In the present study, we performed detailed analysis of mouse sperm flagellar motility to further understand the contribution of glycolysis to mammalian sperm motility. Mouse sperm were able to maintain vigorous motility and produce sufficient ATP in the presence of substrates either for glycolysis or for respiration. However, inhibition of glycolytic enzyme caused a significant decrease in the bend angle of the flagellum, sliding velocity of outer doublet microtubules and intraflagellar ATP content even in the presence of respiratory substrates. The decrease of flagellar bend angle and sliding velocity are prominent at the distal part of the flagellum, indicating that glycolysis inhibition caused ATP deficiency therein. Based on these results, we hypothesized that glycolysis transfer energy synthesized by respiration at the mitochondria located in the basal part of the flagellum to the distal part, by acting as a spatial ATP buffering system. In order to validate this hypothesis, we performed calculation of intraflagellar concentration profiles of adenine nucleotides along the flagellum with or without glycolytic enzyme activity by computer simulation analysis. The result demonstrated that glycolysis has the ability to transfer ATP from mid piece to the tip of the flagellum. It is suggested that glycolysis contributes to energy homeostasis in mouse sperm not only by ATP production, but also by energy transfer.