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 respective proteins were functionally and structurally characterized with the aim of understanding the biological significance of these proteases in Antarctic fish. Both APEH isoforms (APEH-1Tb and APEH-2Tb) showed distinct temperature-kinetic behavior, with significant differences in the Km values. Moreover, beside the typical acylpeptide hydrolase activity, APEH-2Tb showed remarkable oxidized protein endohydrolase activity towards oxidized BSA, suggesting that this isoform could play a homeostatic role in removing oxidatively damaged proteins, sustaining the antioxidant defense systems. The 3D structures 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 showing distinct molecular and temperature–kinetic behaviors. In addition, we suggest that the members of the new cluster ‘APEH-2’ could participate in reactive oxygen species detoxification as phase 3 antioxidant enzymes, enhancing the protein degradation machinery.