The liquid storage of turkey semen without the loss of fertilizing ability is of practical interest to poultry industry. However, fertility rates from liquid stored turkey semen decline within a few hours. A clear cause of the decline in spermatozoa quality is unknown. Therefore, the purpose of the present study was to monitor dynamic of proteomic changes of spermatozoa during liquid storage at three time points (2, 24 and 48h) by 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with a matrix-assisted laser desorption/ionization mass (MALDI TOF/TOF) spectrometry. A total of 57 protein spots were differentially expressed in fresh and stored spermatozoa; 42 spots were more- and 15 were less- abundant in spermatozoa after 48 h of semen storage. Most of the proteins that changed in response to liquid storage were related to i) flagellum dependent cell motility, ii) energy derivation by oxidation of organic compounds and iii) introduction of fertilization, suggesting complexity of the processes leading to decreases of stored semen quality.