<p>Male infertility remains prevalent, with many cases unexplained due to limited understanding of sperm physiology. Capacitation, the process by which ejaculated sperm acquire fertilizing ability within the female reproductive tract, requires coordinated activation of signaling and metabolic pathways. Although glucose, pyruvate, and lactate are present in the tract and routinely included in in vitro capacitation media, an integrative understanding of how each of these substrates contributes to sperm mitochondrial activity and fertilization ability has been lacking. Using the mouse model, we showed that none of the individual substrates was sufficient to induce maximal mitochondrial membrane potential in sperm during capacitation; instead, optimal levels were achieved only when glucose was combined with either pyruvate or lactate. While pyruvate alone sustained progressive motility, it failed to promote hyperactivation and fertilization. Conversely, glucose-only capacitated sperm underwent normal acrosome reaction and hyperactivation but exhibited delayed fertilization. This delay was accompanied by increased oxidative stress, evidenced by elevated reactive oxygen species, lipid peroxidation and DNA fragmentation. Antioxidant supplementation under the glucose-only condition preserved redox balance and fertilization performance. These findings reveal that glucose is not a metabolically neutral substrate; when present alone, it induces oxidative stress that limits sperm function. Importantly, this study highlights the complementary roles of glucose and pyruvate in shaping bioenergetics and redox homeostasis during sperm capacitation. An altered carbon source environment in the female tract may therefore compromise sperm function. These insights have implications for understanding some aspects of unexplained infertility and improving clinical strategies to support fertilization.</p>