Spermatozoa require substantially more ATP than other cells, not only for sustaining sperm motility but also for regulating protein phosphorylation during capacitation. In this study, we have reported for the first time the presence of the two key enzymes of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in ovine spermatozoa by indirect immunofluorescence, Western blotting, in-gel activity, and reverse transcription polymerase chain reaction analysis. We found that the activity of both enzymes significantly increased after in vitro capacitation in the presence of high-cAMP levels, with a concomitant increase in protein tyrosine phosphorylation and in the proportion of sperm-capacitated pattern assessed by the chlortetracycline staining. These results suggest that PPP is related with the progress of capacitation and that a relationship between calcium compartmentalization, protein tyrosine phosphorylation and PPP seems to exist. This is the first report that shows a connection between the PPP, cAMP/PKA signaling pathways and sperm capacitation. These findings can be of high-biological importance to improve our knowledge of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.