The ability of AFPs to protect sperm quality during the freezingthawing procedure has been previously tested in mammals. Few studies have so far been carried out concerning the role of AFPs in fish sperm cryopreservation. Recently, the protective effect of AFPs on plasma membrane lipid composition of sea bream spermatozoa subjected to freezingthawing procedure has been demonstrated. However, studies of AFPs action on protein profile of cryopreserved sperm are lacking, in spite of the fact that defects in sperm proteins may compromise sperm motility, fertilization ability and the early events after fertilization. It has been demonstrated that the alteration of sperm proteins due to the freezing-thawing procedure may contribute to the observed changes in spermatozoa quality. The aim of our work was to evaluate the “protective” effect of the AFPs on sperm proteins during freezing-thawing procedure and, also, to identify specific proteins of flagella and head plasma membranes spermatozoa as potential markers related to the procedures of fish sperm cryopreservation. In particular, we analyzed by 2-DE associated with Nano-LC mass spectrometry the effect of AFPs on the pattern of sperm proteins extracted from isolated flagella and head plasma membranes. Our results show a higher ability of AFPIII, as compared to AFPI, in the protection of sperm proteins during the freezing-thawing procedure. AFPI and AFPIII differently affect the protein profile of the two investigated domains. In the present study we demonstrate that the addition of AFPIII to the extender medium significantly increased, with respect to control, viability, motility rate and VSL of thawed spermatozoa. Moreover, we analysed, for the first time in sperm, the effect of AFPs on the profile of proteins extracted from isolated flagella and heads plasma membranes from sea bream spermatozoa. The results of the 2DE experiments suggest a higher ability of AFPIII, as compared to AFPI, to protect proteins during the freezing-thawing procedure. Studies of threedimensional structures of both AFPs revealed important differences between them. AFPI is a small hydrophobic a-helix, whose direct interaction with phospholipids was suggested by Inglis et al. working with liposomes. AFPIII is a globular protein with several hydrophilic and hydrophobic domains. A direct interaction between AFPIII and the plasma membrane lipids has been indicated in our recent study. In sea bream spermatozoa we showed that the addition of AFPIII to DMSO may avoid changes, which usually occur by freezing with DMSO alone, in membrane phospholipids composition as well as in the saturation/ unsaturation degree of their component fatty acids. Activities and expression of several proteins are well known to be affected by the lipid micro domain surrounding membrane proteins. This aspect should be taken into account when examining the protein expression of sperm cryopreserved in the different experimental conditions tested in the present study, i.e. in DMSO without or with AFPs.