Mammalian sperm are highly polarized in order to accomplish their physiological function, fertilization. Sperm flagellar movement is activated on ejaculation into the extracellular environment. Sperm motility becomes hyperactivated during “capacitation”, which is required for fertilization. For mammalian sperm, ATP supplementation is essential for not only maintaining flagellar motility but also for regulating the phosphorylation events relating motility and fertilization. It was believed that oxidative phosphorylation worked mainly to provide ATP because of sperm morphological characteristics and the efficiency of ATP production. However, recent studies revealed that glycolysis in the principal piece works as the major process for supplying ATP, since glycolysis enzymes are localized to the principal piece, some of them are tightly associated with the cytoskeletal structure in it. We observed that extracellular glucose was highly utilized for the entire flagellar bending motion with a high beat frequency compared with pyruvate, a substrate for respiration. In this review, we discuss how glycolysis regulates sperm motility and function in the mouse.