A 34 kD selenoprotein purified from rat testis was identified as a specific sperm nuclei glutathione peroxidase (snGPx) with similar properties to phospholipid hydroperoxide glutathione peroxidase (PHGPx). The determination of its primary structure by analysis of its first N‐terminal amino acids, database search, polymerase chain reaction, and sequencing of the cDNA showed that it differs from PHGPx in its N‐terminal sequence. This sequence, which is encoded for by an alternative exon in the first intron of the PHGPx gene, shows more than 50% homology to the protamine sequences and contains a nuclear localization signal. In rats, snGPx is highly expressed in the nuclei of the late spermatids where it is the only selenoprotein present. Its appearance coincides with the reorganization of DNA, which leads to highly condensed chromatin stabilized by cross‐linked protamine thiols. In selenium‐depleted rats where the concentration of snGPx had decreased to one‐third of the normal level, chromatin condensation was severely disturbed. We provided evidence that snGPx acts as a protamine thiol peroxidase responsible for disulfide cross‐linking by reduction of reactive oxygen species. Its dual function in chromatin condensation and the protection of sperm DNA against oxidation is necessary to ensure male fertility and sperm quality.