Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique intracellular enzyme that directly reduces lipid hydroperoxides in membranes and has the ability to use protein thiol groups as donor substrates. Three isoforms of PHGPx have so far been identified, namely, a mitochondrial, a cytosolic and a nuclear variant. This article is focused on recent evidence demonstrating that (1) mitochondrial and nuclear PHGPx isoforms display a different pattern of expression during male germ cell differentiation; (2) different PHGPx isoforms play specific and independent functions during sperm maturation. The data are discussed in light of the idea that PHGPx is a moonlighting protein, changing roles depending on the intracellular localization, expression in a specific cell type and different partners which it interacts with.