The cryopreservation of avian semen without significant loss of fertilizing ability is of practical interest to the poultry industry and for the conservation of genetic biodiversity. However, the unique physiological features make avian spermatozoa highly sensitive to damage caused by cryopreservation. The proteomic changes in turkey (Meleagris gallopavo) semen throughout the cryopreservation process have never been investigated previously. Therefore, the purpose of the present study was to evaluate the proteomic changes of extracellular fluid (ECF)of semen during cryopreservation at three stages (fresh, equilibrated, and frozen/thawed semen) using 2-dimensional difference in-gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization mass spectrometry (MALDI TOF/TOF). Six protein spots were significantly enriched in ECF proteome after equilibration compared to fresh semen. Twenty seven protein spots were enriched in ECF after cryopreservation compared to fresh semen. Twenty six protein spots were significantly enriched in ECF proteome after cryopreservation compared to equilibrated semen. The proteins enriched in ECF after cryopreservation were mainly related to NADH metabolic and glycolytic process.