Our previous study using comparative genome analysis revealed a significant gene copy number gain of Dissostichus mawsoni selenoprotein P (Dm-SEPP) during the evolutionary radiation of Antarctic notothenioids, suggesting that Dm-SEPP contribute to this process, but the detailed structure and function of this gene product remain unclear. In the present study, the Dm-SEPP cDNA was cloned and characterized. The Dm-SEPP cDNA contains 17 selenocysteines (Sec) encoded by TGA codons and 2 typical SECIS elements located in the 3'-UTR. Evolutionary analysis of the Dm-SEPP gene revealed that it's closely related to the SEPP gene of zebrafish (Danio rerio), showing 51% amino acid similarity. Over-expression of Dm-SEPP could protect mammalian cells under cold pressure, probably via eliminating ROS. Further study showed an increase of endogenous SEPP in zebrafish ZF4 cells under cold pressure, and knockdown of SEPP decreased cell viability, accompanied with increased ROS. Our results suggested a protective role of Dm-SEPP in cold adaptation in Antarctic notothenioids.