Glutathione peroxidase 4 (Gpx4) is an essential antioxidant enzyme having multiple functions. A long form Gpx4 protein and a short form Gpx4 protein, which are distinguishable by the presence or lack of a mitochondrial signal peptide at the N terminus, are generated from the Gpx4 gene. In this study, we generated transgenic mice using mutated GPX4 genes encoding either the long form Gpx4 (lGPX4 gene) or the short form Gpx4 (sGPX4 gene). Our results showed that transgenic mice with the sGPX4 gene had increased Gpx4 protein in all tissues and were protected against diquat-induced apoptosis in liver. Moreover, the sGPX4 gene was able to rescue the lethal phenotype of the mouse Gpx4-null mutation. In contrast, transgenic mice with the lGPX4 gene had increased Gpx4 protein only in the testes, and the lGPX4 gene failed to rescue the lethal phenotype of the mouse Gpx4-null mutation. In Gpx4-null mice rescued by the sGPX4 gene, the Gpx4 protein was present in mitochondria isolated from somatic tissues, and the submitochondrial distribution pattern of the Gpx4 protein in these mice was identical to that in wild-type mice. Interestingly, the male Gpx4-null mice rescued by the sGPX4 gene were infertile and exhibited sperm malformation. Together, our results demonstrated for the first time that the short form Gpx4 protein is present in somatic tissue mitochondria and is essential for survival and protection against apoptosis in mice, whereas the long form Gpx4 protein is important for male fertility.