Excessive production of reactive oxygen species (ROS) may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Recently, ROS have gained attention as important second messengers. ROS lifetimes can be very short, and many types of ROS cannot penetrate organelle membranes. It is therefore thought that only ROS signal molecules that are generated locally in an organelle are transduced when cells are stimulated. Lipid hydroperoxides are one type of ROS of which the biological function has not yet been clarified. The phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme and separately distributed to the mitochondria, nucleus, nucleoli, and cytosol, where it regulates phospholipid hydroperoxide and fatty acid hydroperoxide as signal molecules. This review focuses on the structure and biological functions of PHGPx in mammalian cells. Overexpression of different types of PHGPx in the RBL2H3 cell line provides a useful model system with which to clarify the ability of different types of PHGPx to modulate cellular function and the importance of lipid hydroperoxides as signal molecules. Transformant studies show that lipid hydroperoxide is an activator of lipoxygenase and cyclooxygenase and participates in inflammation, cardiolipin hydroperoxide is the signal molecule for the release of cytochrome c during apoptotic cell death, and PHGPx is a signal regulator in the IgE receptor-mediated signaling pathway. It is becoming clear that PHGPx has an important role in spermatogenesis, sperm function, and embryonic development, and its deficiency is implicated in human infertility and in embryonic lethality of PHGPx knockout mice.