Updated project metadata. Hormonal stimulation in common carp is a routine practice to enhance sperm production and control gamete maturation. The mechanism regulating functional changes of semen composition upon hormone treatment is unknown at present. The aim of the study was to compare proteome of carp spermatozoa and seminal plasma from control (PBS, n=10) and hormonally stimulated males (D-Ala6,Pro9-NEt)-mGnRH+metoclopramide, n=10). Comparative proteomics based on two-dimensional differential in-gel electrophoresis was employed to quantitatively analyze the spermatozoa and seminal plasma proteins changed in abundance due to hormonal stimulation. Changes in phosphorylation and oxidation level of spermatozoa proteins were also analyzed. The proteomic results were validated by western blot, enzymatic assay and mRNA expression analysis.In total we identified 62 spots (38 up and 24 down regulated) corresponding to 48 proteins with differential abundance in spermatozoa and 36 spots (23 up and 13 down regulated) corresponding to 23 proteins differentially abundant in seminal plasma upon hormone treatment (P<0.05; fold change 1.2). The majority of spermatozoa proteins changed in abundance as well as phosphorylation and carbonylation level were involved in cillium assembly and organization, energy metabolism, protein folding, and signal transduction while differentially abundant seminal plasma proteins were associated with immune and stress response, lipid metabolism and tissue remodelling. Our results strongly suggest that hormonal treatment is not only related to hydration of testis but also induces modification of spermatozoa proteins and synthesis of seminal plasma proteins. ROS signaling and cAMP/PKA and calcium/calmodulin pathways are involved in sperm response to hormonal stimulation.