In order to fertilize the egg, spermatozoa must undergo a series of biochemical processes in the female reproductive tract collectively called capacitation. Only capacitated sperm can interact with the egg resulting in the acrosome reaction (AR), allowing egg penetration and fertilization. Sperm can undergo spontaneous AR (sAR) before reaching the egg, preventing successful fertilization. Here we investigated the metabolic pathways involved in sperm capacitation and sAR. Inhibition of glycolysis or oxidative phosphorylation did not affect capacitation or sAR levels; however, when both systems were inhibited, no capacitation occurred, and there was a significant increase in sAR. Under such ATP-starvation, the increase in sAR is triggered by Ca2+ influx into the sperm via the CatSper cation channel. Protein kinase A (PKA) is an essential key enzyme in sperm capacitation; there was no change in its activity when a single metabolic system was inhibited, while complete inhibition of was observed when the two systems were inhibited. Protein tyrosine phosphorylation (PTP), also known to occur in sperm capacitation, was partially reduced by inhibition of one metabolic system, and completely blocked when the two metabolic systems were inhibited. We conclude that ATP, PKA and PTP are involved in the mechanisms protecting sperm from sAR.