Phospholipid-hydroperoxide glutathione peroxidase (PHGPx), a ubiquitous antioxidant enzyme in the glutathione peroxidase (GPx) family, plays multiple roles in different organisms. Here, a novel PHGPx (PmPHGPx) was identified from Penaeus monodon. The full-length PmPHGPx cDNA was 1885 bp in length with a 489-bp open reading frame (ORF) containing a selenocysteine codon, TGA177−179, and a selenocysteine insertion sequence in the 3′-UTR. The typical signature motifs of the GPx family were also detected in the PmPHGPx amino acid sequence. The PmPHGPx expression pattern showed tissue-specific variations, with the highest expression level in the heart and the lowest expression level in the muscle. To examine the relationship between Pmp53 and PmPHGPx, Pmp53 was successfully silenced with a dsRNA-p53 injection, and an obvious down-regulation in PmPHGPx expression was apparent. To clarify the functional roles of Pmp53 and PmPHGPx, their expression patterns were also assessed after pH-induced stress, salinity stress and heavy metal (Cu, Zn, and Cd) challenges. Similar trends in the expression profiles for PmPHGPx and Pmp53 were detected in both the gills and hepatopancreas in response to all stressors. Therefore, we conclude from the results that PmPHGPx acts synergistically and subsequently works cooperatively with Pmp53 toward mediating cell stress. This study improves our understanding of PmPHGPx and its synergistic role with Pmp53 in counteracting stressors in P. monodon.