The role of reactive oxygen species (ROS) as signal transduction elements in physiological phenomena is a recent concept that changes the paradigm of these active species as harmful molecules that promote deleterious effects and even cell death. Capacitation is a term used to define a complex and not well-characterized process that allows spermatozoa to complete their preparation to fertilize oocytes. Spermatozoa from many species incubated under specific conditions have the ability to produce small amounts of ROS without harming cell function and rather promoting signal transduction pathways associated with capacitation. This review summarizes the findings regarding the role of ROS during mammalian sperm capacitation, specifically as physiological mediators that trigger phosphorylation events. The role of ROS as regulators of protein tyrosine phosphorylation has been known for a decade but other novel phosphorylations, such as those of PKA substrates, of MEK-like proteins, and of proteins with the threonine–glutamine–tyrosine motif, were recently evidenced. Here we stress the involvement of PKA and the ERK pathway as two signal mechanisms acting independently that contribute to the modulation of protein tyrosine phosphorylation required for spermatozoa to achieve capacitation. Moreover, integration of all these data reinforces the concept that although some phosphorylation events are independent of the others, cross talk is also needed among the various pathways involved.