Oxidative challenge is an important factor affecting the adaptive strategies of Antarctic fish, but data on antioxidant defenses in these organisms remain scarce. In this context, a key role could be played by Acylpeptide hydrolase (APEH), which was recently hypothesized to participate in the degradation of oxidized and cytotoxic proteins, although its physiological function is still not fully clarified. This study represents the first report on piscine members of this enzyme family, specifically from the Antarctic teleost Trematomus bernacchii. The cDNAs corresponding to two apeh genes were isolated and the relative proteins were functionally and structurally characterized with the aim to understand the biological significance of these proteases in the Antarctic fish. Both APEH isoforms (APEH-1Tb and APEH-2Tb ) displayed a distinct temperature-kinetic behavior with significant differences in the Km values. Moreover, beside the typical acylaminoacyl peptidase activity, APEH-2Tb showed a remarkable Oxidized Protein Endo-Hydrolase (OPEH) activity towards oxidized BSA, suggesting that this isoform could play a homeostatic role in removing oxidatively damaged proteins, sustaining the antioxidant defense systems. The structural 3D models of both APEHs were predicted and a possible relationship was found between the substrate specificity/affinity and the marked changes in the number of charged residues and hydrophobicity properties surrounding their catalytic sites. Our results demonstrated the occurrence of two APEH isoforms in T. bernacchii, belonging to different phylogenetic clusters identified for the first time and displaying distinct molecular and temperature-kinetic behaviors. In addition, we suggest that the members of the new cluster "APEH-2" could participate in ROS detoxification as Phase3 antioxidant enzymes, enhancing the protein degradation machinery. This article is protected by copyright. All rights reserved.